Neuron generation, regeneration and protection

ABSTRACT

The invention demonstrates that, contrary to apoptotic rabies virus G proteins, certain non-apoptotic rabies virus G proteins, such as the G protein of the CVS-NIV strain, have a neurite outgrowth promoting effect. The invention further demonstrates that this neurite outgrowth promoting effect is due to the cytoplasmic tail of the non-apoptotic rabies virus G proteins, more particularly to their PDZ-BS, which shows a single-point mutation compared to the one of apoptotic rabies virus G proteins. The invention provides methods for inducing and/or stimulating neurite outgrowth, which are useful in inducing neuron differentiation, for example for the treatment of a neoplasm of the nervous system, as well as in regenerating impaired neurons, for example for the treatment of a neurodegenerative disease, disorder or condition or in the treatment of a microbial infection, or in protecting neurons from neurotoxic agents or oxidative stress.

CROSS REFERENCE TO RELATED APPLICATIONS

This application is a divisional of U.S. application Ser. No. 13/263,050filed Oct. 5, 2011, which is a 371 National Stage Application ofPCT/IB2010/000967 filed Apr. 2, 2010, which claims priority to EuropeanApplication No. 09290257.6 filed Apr. 7, 2009, all of which areincorporated by reference in their entireties.

BACKGROUND

1. Field of the Invention

The invention relates to neuron generation, regeneration and protection,more particularly to neurite outgrowth.

2. Description of Related Art

The invention provides means to induce and/or stimulate neuritogeniceffects, which are useful in the medical field, more particularly in thetreatment and/or palliation and/or prevention of disorders of thenervous system. More particularly, the invention provides means forinducing and/or stimulating neurite outgrowth, which are useful forinducing neuron differentiation, for example for the treatment of aneoplasm of the nervous system, as well as in regenerating impairedneurons, for example for the treatment of a neurodegenerative disease,disorder or condition or in the treatment of a microbial infection, orin protecting neurons from neurotoxic agents or oxidative stress.

The means of the invention are based on certain non-apoptotic rabiesvirus G proteins, more particularly on the cytoplasmic tail thereof.

During development of the nervous system, neurons extend axons overconsiderable distances in order to innervate their targets in anappropriate manner. This involves the stimulation in the cells ofspecific signaling pathways which can stimulate the activity of thegrowth cone.

While the developing nervous system, more particularly the developingcentral nervous system, is highly plastic, the adult nervous system,more particularly the adult brain, has more limited repair potential.Therefore, neurite-axon outgrowth and protection against degenerationare important factors to be considered to improve the outcome of aneurodegenerative disease, disorder or condition, such as an acuteinjury of the nervous system or a chronic neurodegenerative disorder.Products, which would be capable of inducing neurite outgrowth from suchneuronal cells, would bring a very useful therapeutic and/or preventiveand/or palliative solution to such diseases, disorders or conditions.

At the other side of the neuron developmental process, the proliferationof neuronal progenitors, which do not differentiate into maturedneuronal structures, leads to nervous system neoplasm. Products, whichwould be capable of inducing neurite outgrowth from such progenic cells,would bring a therapeutic and/or preventive and/or palliative solutionto such neoplasms.

The general idea on the infection of neuronal cells by a neurotropicvirus is that it has no positive impact on neuron morphology, moreparticularly on neurite outgrowth.

Indeed, there are numerous examples showing that neurotropic virusescause neuronal cell death by apoptosis. This concerns both DNA viruses,such as herpes viruses, and RNA viruses, either enveloped such asalphaviruses, bunyaviruses and paramyxoviruses, or unenveloped such aspicornaviruses and reoviruses.

Rabies virus, more particularly attenuated rabies virus strains, hasalso been described as inducing neuronal apoptosis.

For example, WO 03/048198 relates to rabies virus G proteins andfragments thereof of at least 100 amino acids, which induce thedisruption of the neuronal cell integrity and the formation of apoptoticbodies. These apoptotic bodies are capable of stimulating a humoralimmune response, preferably a B-dependent humoral immune response. WO03/048198 shows that:

-   -   attenuated rabies virus strain (such as the attenuated ERA        strain) induces the apoptotic rupture of the cells it infects,    -   the apoptotic bodies thereby produced stimulate a humoral immune        response, more particularly a B-dependent humoral immune        response;    -   induction of apoptosis by a rabies virus strain is determined by        the nature of its G-protein;    -   a rabies virus containing the G protein from an attenuated        rabies virus strain (such as the attenuated ERA strain) is able        to trigger apoptosis of human cells, whereas expression of the G        protein from a pathogenic rabies virus (such as a Challenge        Virus Standard -CVS- strain) is not (cf. more particularly        example 5 and FIGS. 19 and 20 of WO 03/048198).        Please see also Lay et al. 2003 and Préhaud et al. 2003.

Thus, the G proteins of apoptotic rabies virus strains, such as the Gprotein of the attenuated ERA strain, are known to be useful instimulating a humoral response, more particularly a B-dependent humoralimmune response.

Since these particular G proteins induce the apoptosis of the cells theyinfect, they have also been proposed as candidate agents to eliminateundesirable cells by apoptotic rupture of the target cells.

It has also been described that the pathogenicity of a rabies virusstrain is inversely correlated with its ability to induce apoptosis (cf.WO 03/048198; Ugolini 1995; Sarmento et al. 2005; Ugolini 2008; Jacksonet al. 2008).

Therefore, the more virulent a rabies virus strain is, the lessapoptotic.

The findings that virulent rabies virus strains, such as CVS strains, donot induce neuron apoptosis and thereby escape humoral detection explainwhy virulent rabies virus strains can propagate so extensively withinthe CNS before the appearance of signs and symptoms of the disease.

Further studies have been conducted to analyze the changes in geneexpression pattern that are induced upon infection by a neurotropicvirus, such as a rabies virus or herpes simplex type 1 (HSV-1).

These studies have brought the demonstration that post-mitotic humanneurons, in the absence of glia, have the intrinsic machinery to sensevirus infection, and that neurotropic viruses, such pathogenic rabiesvirus or HSV-1, induce the release of cytokines from post-mitotic humanneurons (Préhaud et al. 2005). This cytokine release is believed tofurther contribute to the escape of neurons from apoptosis and in theconsequent spreading of such neurotropic viruses.

Therefore, neuronal cell death mechanisms, as well as the capacity ofneurons to raise an immune response upon viral infection, have beenthoroughly investigated.

However, less is known about the processes involved in neurogeneration,neuroregeneration and neuroprotection, more particularly in neuriteoutgrowth from pre-mitotic neurons, such as neuronal progenitors orneoplastic neurons, or from degenerative neurons.

If one focuses on rabies virus, a schematic summary of the currentknowledge would be that attenuated rabies virus strains are known tohave medical applications due to the apoptosis they induce, and thatvirulent rabies virus strains are known to not induce apoptosis, but, tothe contrary, to preserve the neuronal network, which favors theirspreading.

While virulent rabies virus strains have been described as preservingthe integrity of the neuronal network, they have also been reported ashaving a negative impact, or at the very least no positive impact, onneuronal morphology, more particularly on neurite outgrowth.

For example, the publication Guigoni and Coulon 2002 describes that thevirulent rabies virus strain CVS-Gif-sur-Yvette do not induce neuriteoutgrowth from rat motoneurons (cf. for example, FIGS. 5A and 5B of thispublication).

Negative impact on neurite outgrowth has also been reported. Forexample, the publication Scott et al., 2008 has reported that thepathogenic CVS-11 rabies virus strain induces beading of the dendritesand axons, i.e., the formation of vacuoles that are characteristic of anegative stress impact.

The invention provides means for the generation, regeneration andprotection of neurons, which derive from certain pathogenic rabies virusstrains, and which show surprising and unexpected properties.

SUMMARY

The invention demonstrates that some pathogenic (and non-apoptotic)rabies virus G proteins have a neurite outgrowth promoting effect, i.e.,that some non-apoptotic rabies virus G proteins induce and/or stimulateneuritogenesis. This effect is not shown by apoptotic rabies virus Gproteins. Neither is it shown by all non-apoptotic rabies virus Gproteins.

The inventors identified a sub-group of non-apoptotic (and virulent)rabies virus strains, the G protein of which has a significantlypositive effect on neurite outgrowth. As described and illustratedbelow, a representative strain of this sub-group is the CVS-NIV strain.The G protein of this non-apoptotic (and virulent) strain differs byonly 6 amino acid from the G protein of the apoptotic (and attenuated)ERA strain (CNCM I-2760).

The invention further demonstrates that this neurite outgrowth promotingeffect is due to the cytoplasmic tail of said non-apoptotic rabies virusG proteins, more particularly to their PDZ binding site (PDZ-BS) and/orto an amino acid which, in the sequence of the G protein of the CVS-NIVstrain, is at position 491, more particularly to their PDZ-BS.

The invention relates to polypeptides, which are or derive from certainnon-apoptotic rabies virus G proteins, more particularly from theircytoplasmic tail, as well as to nucleic acids, vectors, cells andpharmaceutical compositions or drugs.

The means of the invention are notably useful as neurite outgrowthstimulating and/or inducing agents. They can notably be used forinducing neuron differentiation, for example in the treatment of aneoplasm of the nervous system, as well as for regenerating impairedneurons, for example in the treatment of a neurodegenerative disease,disorder or condition or in the treatment of a microbial infection, orfor protecting neurons from neurotoxic agents or oxidative stress.

BRIEF DESCRIPTION OF THE DRAWINGS

Some of the figures, to which the present application refers, are incolor. The application as filed contains the color print-out of thefigures, which can therefore be accessed by inspection of the file ofthe application at the patent office.

FIG. 1: neurogeneration, neuroregeneration and neuroprotection areimplicated in various neuronal disorders, conditions and diseases,including neurotoxicity, seizure, stroke, trauma, aging,neurodegenerative disease, encephalopathy.

FIG. 2: schematic structure of the recombinant rabies virus (rRABV)produced by the inventors and used in the examples below.

The G protein of the CVS-NIV strain (non-apoptotic strain) differs byonly 6 aa from the G protein of the ERA strain (apoptotic strain).

G-survival=G protein of the CVS-NIV strain

G-death=G protein of the ERA strain

G-cyto death=cytoplasmic tail of G-death in a CVS-NIV G gene background

G-cyto survival=cytoplasmic tail of G-survival in an ERA G genebackground

SP=signal peptide

EC: extracellular domain

TM: transmembrane domain

Cyto: cytoplasmic domain

FIGS. 3A, 3B, 3C, 3D: the cytoplasmic tail of the G protein of theCVS-NIV strain (non-apoptotic strain) contains a molecular signaturepromoting neurite outgrowth.

FIG. 3A: neurite outgrowth assay with rRABV G-CVS-NIV and rRABV G-ERA

FIG. 3B: results of the neurite outgrowth assay with db-cAMP with rRABVG-CVS-NIV and rRABV G-ERA

FIG. 3C: results of the neurite outgrowth assay without db-cAMP withrRABV G-CVS-NIV, rRABV G-CVS-Cyto Death, rRABV G-ERA and rRABVG-ERA-Cyto Survival

FIG. 3D: results of the neurite outgrowth assay with rRABV-G-CVS-NIV,rRABV G-CVS (LQ) and rRABV G-CVS (HE)

N.I.=non infected

rRABV=recombinant rabies virus

G-CVS or G-CVS-NIV=protein G of the CVS-NIV strain

G-CVS-Cyto Death=cytoplasmic tail of the G protein of the ERA strain ina CVS-NIV G gene background

G-ERA=protein G of the ERA strain

G-ERA-Cyto Survival=cytoplasmic tail of the G protein of the CVS-NIVstrain in an ERA G gene background

rRABV G-CVS (LQ)=recombinant rabies virus G protein of the CVS-NIVstrain, wherein the amino acid H, which is at position 491 in the fulllength G protein of the CVS-NIV strain, has been replaced by the aminoacid L

rRABV G-CVS (HE)=recombinant rabies virus G protein of the CVS-NIVstrain, wherein the amino acid Q, which is at position 521 in the fulllength G protein of the CVS-NIV strain, has been replaced by the aminoacid E

FIGS. 4A, 4B: the cytoplasmic tail of the G protein of the CVS-NIVstrain (non-apoptotic strain) is an intrinsic effector promotingneuritogenesis, which works synergistically with cAMP*

FIG. 4A: without db-cAMP

FIG. 4B: without db-cAMP (first two histograms) and with cAMP (last twohistograms)

★ means significantly different (Student's t test with p=0.0067)

N.I.=non infected

rRABV=recombinant rabies virus

G-CVS or G-CVS-NIV=protein G of the CVS-NIV strain

G-CVS-Cyto Death=cytoplasmic tail of the G protein of the ERA strain ina CVS-NIV G gene background

G-ERA=protein G of the ERA strain

G-ERA-Cyto Survival=cytoplasmic tail of the G protein of the CVS-NIVstrain in an

ERA G gene background

FIG. 5: the neuritogenesis effect of the cytoplasmic tail of the Gprotein of the CVS-NIV strain (non-apoptotic strain) is dependent on themolecular signature and not on the amount of expressed G protein.

rRABV=recombinant rabies virus

G-CVS or G-CVS-NIV=protein G of the CVS-NIV strain

G-CVS-Cyto Death=cytoplasmic tail of the G protein of the ERA strain ina CVS-NIV G gene background

G-ERA=protein G of the ERA strain

G-ERA-Cyto Survival=cytoplasmic tail of the G protein of the CVS-NIVstrain in an

ERA G gene background

FIGS. 6A, 6B: the cytoplasmic tail of the G protein of the CVS-NIVstrain (non-apoptotic strain) confers neuroprotection against growthcone collapsing drug (LPA)

FIG. 6A: with db-cAMP

FIG. 6B: without db-cAMP

N.I.=non infected

rRABV=recombinant rabies virus

G-CVS or G-CVS-NIV=protein G of the CVS-NIV strain

G-CVS-Cyto Death=cytoplasmic tail of the G protein of the ERA strain ina CVS G gene background

G-ERA=protein G of the ERA strain

G-ERA-Cyto Survival=cytoplasmic tail of the G protein of the CVS-NIVstrain in an

ERA G gene background

FIG. 7: the neuroprotection against the growth cone collapsing drug LPAthat is induced by the cytoplasmic tail of the G protein of the CVS-NIVstrain (non-apoptotic strain) is a robust one.

★ means significantly different (ANOVA test)

▴ A means not significantly different (ANOVA test)

N.I.=non infected

rRABV=recombinant rabies virus

G-CVS-NIV=protein G of the CVS-NIV strain

G-CVS-Cyto Death=cytoplasmic tail of the G protein of the ERA strain ina CVS-NIV G gene background

G-ERA=protein G of the ERA strain

G-ERA-Cyto Survival=cytoplasmic tail of the G protein of the CVS-NIVstrain in an

ERA G gene background

LPA=lysophosphatidic acid

FIG. 8: the cytoplasmic tail of the G protein of the CVS-NIV strain(non-apoptotic strain) confers neuroprotection against oxidative stress(H₂O₂). These experiments were performed without db-cAMP.

★ means significantly different (Student's t test)

▴ means not significantly different (Student's t test)

N.I.=non infected

rRABV=recombinant rabies virus

G-CVS or G-CVS-NIV=protein G of the CVS-NIV strain

G-CVS-Cyto Death=cytoplasmic tail of the G protein of the ERA strain ina CVS-NIV G gene background

G-ERA=protein G of the ERA strain

G-ERA-Cyto Survival=cytoplasmic tail of the G protein of the CVS-NIVstrain in an

ERA G gene background

FIGS. 9A, 9B, 9C: the cytoplasmic tail of the G protein of the CVS-NIVstrain (non-apoptotic strain) confers protection against Herpes VirusSimplex type 1 (HSV-1) cytopathic effect.

N.I.=non infected

rRABV=recombinant rabies virus

G-CVS or G-CVS-NIV=protein G of the CVS-NIV strain

G-CVS-Cyto Death=cytoplasmic tail of the G protein of the ERA strain ina CVS-NIV G gene background

G-ERA=protein G of the ERA strain

G-ERA-Cyto Survival=cytoplasmic tail of the G protein of the CVS-NIVstrain in an

ERA G gene background

FIGS. 10A, 10B: cell proliferation of human neuroblastoma cells treatedwith the all-trans-retinoic acid (ATRA) pro-differentiative drug (FIG.10A: flow cytometry; FIG. 10B: MTT assay).

N.I.=non infected

FIGS. 11A, 11B: the cytoplasmic tail of the G protein of the CVS-NIVstrain (non-apoptotic strain) confers anti-proliferative properties:cell proliferation (FIG. 11A) and neurite length (FIG. 11B) of humanneuroblastoma cells treated with rRABV G-CVS-NIV or rRABV G-ERA

N.I.=non infected

rRABV=recombinant rabies virus

G-CVS-NIV=protein G of the CVS-NIV strain

G-ERA=protein G of the ERA strain

FIGS. 12A, 12B: the cytoplasmic tail of the G protein of the CVS-NIVstrain (non-apoptotic strain) confers anti-proliferative properties:cell proliferation of human neuroblastoma cells treated with rRABVG-CVS, rRABV G-ERA, rRABV G-CVS-Cyto death or rRABV G-ERA-Cyto survival(FIG. 12A: flow cytometry; FIG. 12B: MTT assay) (N.I.=non infected)

N.I.=non infected

rRABV=recombinant rabies virus

G-CVS or G-CVS-NIV=protein G of the CVS-NIV strain

G-CVS-Cyto Death=cytoplasmic tail of the G protein of the ERA strain ina CVS-NIV G gene background

G-ERA=protein G of the ERA strain

G-ERA-Cyto Survival=cytoplasmic tail of the G protein of the CVS-NIVstrain in an

ERA G gene background

FIGS. 13A, 13B: nucleic acid and protein sequences of the G protein ofthe CVS-NIV strain (FIG. 13A) and of the ERA strain (FIG. 13B). In FIG.13A, the PDZ-BS motif of the G protein of the CVS-NIV strain isunderlined (QTRL).

aa=amino acid

FIG. 14: sequence alignment of the G proteins of the CVS-NIV and ERAstrains; the G proteins differ by only 6 amino acids (shown in bold inFIG. 14):

TABLE 1 Position of the amino acid G protein of the G protein of the inthe sequence of the full CVS-NIV strain ERA strain length G protein(non-apoptotic strain) (apoptotic strain) 48 V I 139 H R 179 P S 219 A V491 H L 521 Q E

The alignment shown in FIG. 14 has been performed using the followingparameters:

Comparison matrix: BLOSUM62

Number of alignments computed: 20

Gap open penalty: 12

Gap extension penalty: 4

The result of this alignment is:

98.9% identity in 524 residues overlap;

Score: 2787.0;

Gap frequency: 0.0%.

FIG. 15: nucleic acid and protein sequences of the cytoplasmic domain ofthe G protein of the CVS-NIV strain and of the ERA strain. In FIG. 15,the PDZ-BS motif of the G protein of the CVS-NIV strain is underlined(QTRL).

aa=amino acid

FIG. 16: nucleic acid and protein sequences of the PDZ-BS of the Gprotein of the CVS-NIV strain and of the ERA strain.

aa=amino acid

FIG. 17: alignment of the G protein of the CVS-NIV strain (SEQ ID NO: 2)and of the G protein of the CVS-Gif-sur-Yvette strain (SEQ ID NO: 15).

FIG. 18: alignment of the G protein of the CVS-NIV strain (SEQ ID NO: 2)and of the G proteins of three CVS-11 strains (SEQ ID NO: 16; SEQ ID NO:17; SEQ ID NO: 18).

FIG. 19: sequence of the cytoplasmic fragment of the G protein of theCVS-NIV strain (SEQ ID NO: 6) and of two conservative variants derivingtherefrom (variant sequence A of SEQ ID NO: 19; variant sequence B ofSEQ ID NO: 20).

FIG. 20: the deletion of the PDZ-BS affects the neurite outgrowthphenotype (average neurite length in μm at 8 h post-infection, in thepresence of db-cAMP).

N.I.=non infected;

G-CVS-NIV=protein G of the CVS-NIV strain;

G-CVS-NIV-DeltaPDZ-BS=protein G of the CVS-NIV strain from which thePDZ-BS has been deleted.

FIG. 21: schematic structure of single-point mutants of the invention(variants B and A). Mutant viruses CVS HE (I-4143) and CVS LQ (I-4142)of the invention differ from CVS-NIV (I-4140) by their G proteins, inwhich a single-point mutation has been introduced.

CVS HE (variant B)=single-point mutation of the first PDZ-BS amino acidof the G protein CVS-NIV strain, which is E in CVS HE (position 521)instead of Q.

CVS LQ (variant A)=single-point mutation of the amino acid at position491, which is E instead of Q.

SP: signal peptide

EC: extracellular domain

TM: transmembrane domain

Cyto: cytoplasmic domain

FIG. 22: Amino Acid Sequences of Domains of the G Protein of aNon-Apoptotic Virus Strain and of Construct Inserts

Sequences of G-CVS Domains:

-   -   G full length (SEQ ID NO: 2): full-length G protein of an        apoptotic rabies virus strain (CVS-NIV);    -   Signal peptide (SP; SEQ ID NO: 21): signal peptide of an        apoptotic rabies virus strain (CVS-NIV);    -   Ectodomain (EC; SEQ ID NO: 22): ectodomain of an apoptotic        rabies virus strain (CVS-NIV);    -   Transmembrane domain (TM. SEQ ID NO: 23): transmembrane domain        of an apoptotic rabies virus strain (CVS-NIV);    -   Cytoplasmic domain (Cyto; SEQ ID NO: 6): cytoplasmic domain of        an apoptotic rabies virus strain (CVS-NIV).

Amino Acid Sequences Coded by Construct Inserts:

-   -   Amino acid M+SEQ ID NO: 6=SEQ ID NO: 24 (construct G-Cyto);    -   Transmembrane domain of SEQ ID NO: 23+cytoplasmic domain of SEQ        ID NO: 6=SEQ ID NO: 25;    -   Two amino acids from the C-terminal end of the ectodomain of        G-CVS-NIV (amino acids GK)+transmembrane domain of SEQ ID NO:        23+cytoplasmic domain of SEQ ID NO: 6=SEQ ID NO: 26;    -   Signal peptide of SEQ ID NO: 21+two amino acids from the        C-terminal end of the ectodomain of G-CVS-NIV (amino acids        GK)+transmembrane domain of SEQ ID NO: 23+cytoplasmic domain of        SEQ ID NO: 6=SEQ ID NO: 27 (construct G-(SP-[2a]-TM-Cyto).

FIG. 23: alignment of the amino acid sequences coded by the inserts ofthree constructs (SEQ ID NO: 2 coded by the G-full construct; SEQ ID NO:27 coded by the G-(SP)-[2a]-TM-Cyto (=GSP2aaTMCyto) construct; SEQ IDNO: 24 coded by the G-Cyto construct).

FIG. 24: schematic representation of three RABV G gene constructs(G-full of SEQ ID NO: 2 (=G survival); G-Cyto of SEQ ID NO: 24 (=Gsurvival-Cyto); G-(SP)-[2a]-TM-Cyto of SEQ ID NO: 27 (=G Survival-ΔEC).

FIG. 25: RABV G expression (arbitrary units) for control plasmid, G-Fullcontruct (SEQ ID NO: 2), G-(SP-[2a]-TM-Cyto) construct (SEQ ID NO: 27),and G-Cyto construct (SEQ ID NO: 24) in the SH-SY5Y cell line (humanneurobastoma cell line).

FIGS. 26 and 27: effect on neurite outgrowth of the expression:

-   -   of the full length G protein of a non-apoptotic rabies virus        strain (SEQ ID NO: 2; G-Full contruct),    -   of the transmembrane and cytoplasmic domains of a non-apoptotic        rabies virus strain (SEQ ID NO: 27; G-(SP-[2a]-TM-Cyto)        construct), or    -   of the cytoplasmic domain of a non-apoptotic rabies virus strain        (SEQ ID NO: 24; G-Cyto construct),        in human neuroblastoma cells (SH-SY5Y cell line in the presence        of db c-AMP), compared to control (no DNA) and to the control        plasmid.

FIG. 26: transient expression.

FIG. 27: stable expression.

FIG. 28: the expression of the transmembrane and cytoplasmic domains ofa non-apoptotic rabies virus strain (SEQ ID NO: 27; constructG-(SP-[2a]-TM-Cyto) induces neurite outgrowth from human neuroblastomacells (SH-SY5Y cell line in the presence of db c-AMP), and confersprotection against the growth cone collapsing drug LPA to the neuritesthat have grown.

FIGS. 29 and 30: the (stable) expression of the cytoplasmic domain of anon-apoptotic rabies virus strain induces and/or stimulates thedifferentiation of an embryonic carcinoma cell line (NTera 2cl.-D1; ATCCCRL-1973) into mature post-mitotic human neurons (5 days postdifferentiation procedure: effect of the G-Full construct-insert of SEQID NO: 2-, of the G-(SP-[2a]-TM-Cyto) construct-insert of SEQ ID NO:27-, or of the G-Cyto construct-insert of SEQ ID NO: 24-, compared tocontrol plasmid).

FIG. 29: colorized images.

FIG. 30: neurite tips.

FIG. 31: the (stable) expression of the cytoplasmic domain of anon-apoptotic rabies virus strain induces and/or stimulates thedifferentiation of an embryonic carcinoma cell line (NTera 2cl.-D1; ATCCCRL-1973) into mature post-mitotic human neurons, and induces and/orstimulates the organisation of a neuronal network with long axons(colorized images of live neurons, 50 days post differentiationprocedure: effect of the G-Full construct-insert of SEQ ID NO: 2-, ofthe G-(SP-[2a]-TM-Cyto) construct-insert of SEQ ID NO: 27-, or of theG-Cyto construct-insert of SEQ ID NO: 24-, compared to control plasmid).

FIGS. 32 and 33: expression of the transmembrane and cytoplasmic domainsof a non-apoptotic rabies virus strain induces and/or stimulates theregeneration of wounded mature post-mitotic human neurons (neuronsseeded on PDL-laminin plastic ware, 3 days post scratching withinjection needle (26GX1/2″, 12-4.5); effect of the polypeptide of SEQ IDNO: 27 (construct G-(SP-[2a]-TM-Cyto)-neurite regeneration-, compared tocontrol plasmid-neurite degeneration-).

FIG. 32: colorized images

FIG. 33: percentage of neurite regeneration

DETAILED DESCRIPTION OF A PREFERRED EMBODIMENT

The present application relates to the subject-matter as defined in theclaims as filed and as herein described and illustrated.

Pathogenicity of rabies virus, a neurotropic virus causing fatalencephalitis in most mammalian species, correlates with the ability ofthe infected neurons to survive. Attenuation of laboratory strainsobtained in search of candidate live vaccine is always linked to theability of those vaccine strains to trigger cell death.

Subversion of an infected cell by a given virus implies the perturbationof specific signaling pathways.

Rabies virus is an enveloped, bullet-shaped virus belonging to theRhabdoviridae family and the Lyssavirus genus. The viral particleconsists of a membrane composed of host lipids and two viral proteins,the G and M proteins, surrounding a helical nucleocapsid (NC). The NC iscomposed of a viral negative-strand RNA molecule protected by the Nprotein, the P protein, and the RNA-dependent RNA polymerase, the Lprotein. The rabies virus proteins are not synthesized in equal amountsin infected cells and are not present at the same ratios in viralparticles. Indeed, the N, G and M proteins are, in this order, the mostprominent species in virions.

The strategic decision between preservation of the neuronal networkintegrity (favoring rabies virus spreading) and neuronal cell death(favoring immunogenicity) necessarily involves a choice between at leasttwo signaling networks.

The invention brings the demonstration that the cytoplasmic tail of theG protein of rabies virus strain has a crucial role in the command ofthe pro-survival (i.e., preservation of the neuronal network integrity,which favors rabies virus spreading) versus pro-apoptotic (i.e., celldeath, which favors immunogenicity) decision process.

The invention further uncovers unexpected effects of the pro-survivalstrategy. Indeed, the invention shows that the G proteins of somenon-apoptotic (and virulent) rabies virus strains, more particularly ofthe CVS-NIV strain, have a neurite outgrowth promoting effect, i.e.,that these non-apoptotic rabies virus G proteins induce and/or stimulateneuritogenesis.

This effect is not shown by apoptotic rabies virus G proteins, such asthe G proteins of the ERA and other attenuated rabies virus strains.

Furthermore, this effect is not shown by all apoptotic and/or virulentrabies virus G proteins. More particularly, it is not shown by the Gprotein of the CVS-Gif-sur-Yvette strain (Préhaud et al. 1988), althoughthis G protein has a high identity score with the G protein of theCVS-NIV strain (cf. FIG. 17). Said effect is not shown by the G proteinsof the CVS-11 strains (e.g., ACA57830, AAC34683, ABV24348), althoughthese G proteins also have a high identity score with the G protein ofthe CVS-NIV strain (cf. FIG. 18). Neither is it shown by the G proteinsof the N2C or CVS-24 strains. Therefore, not all virulent rabies virusstrains have a G protein, which shows a significantly positive effect onneurite outgrowth.

Hence, the inventors identified a sub-group of non-apoptotic (andvirulent) rabies virus strains, the G protein of which has asignificantly positive effect on neurite outgrowth. A representativestrain of this sub-group is the CVS-NIV strain. These G proteins mayherein be referred to as G proteins of the CVS-NIV type.

The CVS-NIV strain has been deposited at the CNCM on the 1 Apr. 2009under deposit number I-4140.

A plasmid expressing the G protein of the CVS-NIV strain has beendeposited at the CNCM on the 30 Nov. 2001 under deposit number I-2578.

The G protein of the CVS-NIV strain has been described in Préhaud et al.2003.

The sequence of the G protein of the CVS-NIV strain is available underaccession number AF 406694.

A sequence of the G protein of the CVS-NIV strain is the sequence of SEQID NO: 2 shown in FIG. 13A.

A rabies virus G protein is a type I transmembrane glycoprotein thatforms the trimeric spikes of the viral envelope and that is found at themembrane of infected cells. The rabies virus G protein is a 524 aminoacid long protein, which consists of an ectodomain, a transmembranesegment and a 44 amino acid long cytoplasmic domain.

The very last 4 amino acids of this C-terminal domain form a PDZ bindingsite (PDZ-BS). PDZ (PSD-95, Discs Large, ZO-1) domains form globularstructures of 80-100 aa organized into six beta-strands and two alphahelices creating a socket where the C terminal sequence of a partnerprotein could be inserted.

A PDZ binding site (PDZ-BS) is a 4 amino acid sequence; the PDZ-BSsequence is the sequence of SEQ ID NO: 13:x₁-x₂-x₃-x₄, wherein:

-   -   x₁ is any amino acid, and    -   x₂ is T or S or I, and    -   x₃ is any amino acid, and    -   x₄ is L or V.

The invention notably demonstrates that it is the cytoplasmic tail ofsaid non-apoptotic rabies virus G proteins of the CVS-NIV type, which isresponsible for this neurite outgrowth effect.

The invention further demonstrates that this is notably due to thePDZ-BS that is contained in the cytoplasmic tail of said non-apoptoticrabies virus G proteins of the CVS-NIV type.

The PDZ-BS motif of said non-apoptotic rabies virus G proteins of theCVS-NIV type shows a single-point mutation compared to the one ofapoptotic rabies virus G proteins. This single-point mutation concernsthe first PDZ-BS amino acid, which is not an E in non-apoptotic rabiesvirus G proteins.

The invention further demonstrates that the amino acid, which, in thefull length non-apoptotic rabies virus G protein sequence of the CVS-NIVstrain, is at position 491, also contributes to this neurite outgrowtheffect. This amino acid is H in the G protein of the CVS-NIV strain (cf.SEQ ID NO: 2). Position 491 in the full length G protein corresponds toposition 11 in the cytoplasmic fragment of this protein (SEQ ID NO: 6 inFIG. 15).

The inventors demonstrate that the amino acids, which, in the fulllength non-apoptotic rabies virus G protein sequence of the CVS-NIVstrain (SEQ ID NO: 2), are at positions 491 and 521, are both importantfor the neurite outgrowth effect of the invention. More particularly,they show that H (but not L) at position 491 and Q (but not E) atposition 521, both favors the neurite outgrowth effect of the invention.

Mutant virus, which differ from the CVS-NIV strain in that their Gproteins are H491L single-point mutated or Q521E single point mutated,have been constructed and produced by the inventors (CNCM I-4142 andI-4143).

The H491L mutant G protein still has Q at position 521.

The Q521E mutant G protein still has H at position 491.

Both mutant proteins induce a significantly positive neurite outgrowtheffect, although at a lesser extent than the CVS-NIV G protein, whichhas both H at position 491 and Q at position 521.

The cytoplasmic fragments of these mutant G proteins are shown in FIG.19 (SEQ ID NO: 19 and SEQ ID NO: 20).

DEFINITION OF A POLYPEPTIDE OF THE INVENTION

The invention relates to a polypeptide, which is:

-   -   a polypeptide, which comprises:        -   the parent sequence selected from the sequence of SEQ ID NO:            6, the cytoplasmic fragment of the G protein of the strain            I-4140, the cytoplasmic fragment of the G protein produced            by the plasmid I-2578, the cytoplasmic fragment of the G            protein of sequence AF406694, the cytoplasmic fragment of            the G protein of SEQ ID NO: 2,    -   or        -   a conservative variant sequence of said parent sequence,    -   or        -   a conservative fragment of said parent sequence or of said            conservative variant sequence.

Said parent sequences, conservative variant sequences and conservativefragments sequences advantageously have the function of inducing and/orstimulating neurite outgrowth, for example as below illustrated, moreparticularly neurite outgrowth from human pre-mitotic neurons, e.g.,from the human neuroblastoma cell line SH-SY5Y (ATCC CRL-2266; pleasesee example 1 below for illustrative experimental conditions andmaterials).

The invention more particularly relates to said polypeptide, for use instimulating and/or inducing neurite outgrowth, more particularly for usein the treatment and/or palliation and/or prevention of a disease,disorder or condition involving an insufficient or impaired neuriteoutgrowth, more particularly an insufficient or impaired neuritogenesis.

Preferably, the amino acid length of said polypeptide is of less than100 amino acids, more preferably of less than 90 amino acids.

The sequence of the above-mentioned variant proteins can be a sequence,which does not correspond to any known or identifiable rabies virus Gprotein.

Indeed, one of average skill in the art will recognize that startingfrom the sequence of a given rabies virus G protein, one or severalamino acid substitution(s) and/or addition(s) and/or deletion(s) can bemade, while still retaining the capacity of inducing and/or stimulatingneurite outgrowth. Such conservative amino acid substitution(s) and/oraddition(s) and/or deletion(s) are herein encompassed.

Without being bound by theory, each of the following eight groups areusually considered as containing amino acids that are conservativesubstitutions for one another:

1) Alanine (A), Glycine (G);

2) Aspartic acid (D), Glutamic acid (E);

3) Asparagine (N), Glutamine (Q);

4) Arginine (R), Lysine (K);

5) Isoleucine (I), Leucine (L), Methionine (M), Valine (V);

6) Phenylalanine (F), Tyrosine (Y), Tryptophan (W);

7) Serine (S), Threonine (T); and

8) Cysteine (C), Methionine (M).

Therefore, the above-mentioned variant proteins notably encompassproteins, which have been engineered by man, and which differ from saidrabies virus G protein by one or several amino acid substitution(s)and/or addition(s) and/or deletion(s), provided that the resultingvariant protein still has the capacity of inducing and/or stimulatingneurite outgrowth, more particularly neurite outgrowth from humanpre-mitotic neurons, e.g., from the human neuroblastoma cell lineSH-SY5Y (ATCC CRL-2266; please see example 1 below for illustrativeexperimental conditions and materials).

Preferably, said conservative variant sequence is:

-   -   a variant sequence of said parent sequence, which:        -   is of 34 to 54 amino acids,        -   is at least 94% identical to said parent sequence over the            entire length of the shortest of the parent and variant            sequences,        -   comprises a PDZ-BS sequence (preferably the last four            C-terminal amino acids), wherein said PDZ-BS sequence is            x₁-x₂-x₃-x₄, wherein:            -   x₁ is any amino acid except E (preferably Q), and            -   x₂ is T or S or I (preferably not I, more preferably T),                and            -   x₃ is any amino acid (preferably R), and            -   x₄ is L or V (preferably L)                -   (SEQ ID NO: 14),    -   said variant sequence being referred to as variant sequence A,    -   or    -   a variant sequence of said parent sequence, which:        -   is of 44 amino acids,        -   is at least 94% identical to said parent sequence,        -   comprises a PDZ-BS sequence (preferably the last four            C-terminal amino acids), wherein said PDZ-BS sequence is            x₁-x₂-x₃-x₄, wherein:            -   x₁ is E, and            -   x₂ is T or S or I (preferably not I, more preferably T),                and            -   x₃ is any amino acid (preferably R), and            -   x₄ is L or V (preferably L)                -   (SEQ ID NO: 13 with x₁=E), and        -   does not comprise the amino acid L at position 11            (preferably comprises the amino acid H at this position),    -   said variant sequence being referred to as variant sequence B.

Preferably, said sequence identity is of at least 95% (preferably atleast 96%, more preferably at least 97%, still more preferably at least97.5%, even still more preferably at least 98%, most preferably at least98.5%, still most preferably at least 99%).

More preferably, said conservative variant sequence is a variantsequence A.

Preferably, said conservative fragment is a fragment of at least 34amino acids, which has retained the PDZ-BS sequence of said parentsequence or of said variant sequence A or of said variant sequence B,respectively

For the sake of conciseness, the term “polypeptide” is herein intendedas encompassing proteins, and conversely.

This term also encompasses polypeptides (or proteins), which have beenmodified by post-transcriptional modification and/or by syntheticchemistry, e.g., by adjunction of a non-peptidic chemical group and/orby modification of the tertiary structure of the polypeptide, e.g., byacetylation, acylation, hydroxylation, cyclisation, racemisation,phosphorylation, etc., as long as the resulting modified polypeptide hasretained the capacity of inducing and/or stimulating neurite outgrowth,more particularly neurite outgrowth from human pre-mitotic neurons,e.g., from the human neuroblastoma cell line SH-SY5Y (ATCC CRL-2266;please see example 1 below for illustrative experimental conditions andmaterials).

The polypeptide of the invention advantageously is, on and of its own,neurotropic. If required or desired, the polypeptide of the inventioncan nevertheless be coupled to, or fused with an agent that improves itsneurotropicity, more particularly its tropicity for brain neurons, suchas RVG-9R as described by Kumar et al. 2007.

Amino acid sequences and nucleotide sequences are herein given inaccordance with the standard orientation, i.e., from N-terminal end toC-terminal end for amino acid sequences and from 5′-terminal end to3′-terminal end for nucleotide sequences.

According to an embodiment of the invention, the PDZ-BS sequence of saidvariant sequence A is QTRL (SEQ ID NO: 10).

According to an embodiment of the invention, said variant sequence A isof 44 amino acids and has the amino acid H or L, preferably H, atposition 11.

An illustrative variant sequence A, which is of 44 amino acids and whichhas the amino acid L at position 11 is the sequence of SEQ ID NO: 19(cf. FIG. 19).

According to an embodiment of the invention, the PDZ-BS sequence of saidvariant sequence B is ETRL (SEQ ID NO: 12).

According to an embodiment of the invention, said variant sequence B hasthe amino acid H at position 11.

An illustrative variant sequence B, which is of 44 amino acids and whichhas the amino acid H at position 11 is the sequence of SEQ ID NO: 20(cf. FIG. 19).

According to an embodiment of the invention, said fragment is thefragment 11-44 from the sequence of SEQ ID NO: 6.

A polypeptide of the invention may comprise, or consist of saidpro-neurite outgrowth sequence and of the transmembranar part of a Grabies virus protein or any other anchoring means that the person ofaverage skill in the art may find appropriate to anchor the polypeptideon or in the membrane of a cell, such as a cell as below-defined.

A polypeptide of the invention can be inserted into the structure of anengineered antibody, for example a single chain antibody.

According to an embodiment of the invention, said polypeptide is the Gprotein of a rabies virus strain, or a cytoplasmic fragment thereof(e.g., SEQ ID NO: 6), or a sub-fragment of such a cytoplasmic fragment(e.g., fragment 11-54 of SEQ ID NO: 6). Said rabies virus G protein morepreferably is a non-apoptotic rabies virus G protein. For example, saidrabies virus strain is the strain deposited at the CNCM under I-4140.

According to an embodiment of the invention, said polypeptide comprises,or consists of, a variant sequence A or B (as above-defined) of such acytoplasmic fragment or sub-fragment. For example, said polypeptidecomprises, or consist of, the cytoplasmic fragment of the G protein ofthe rabies virus strain deposited at the CNCM under I-4142 or I-4143(cf. FIG. 21), whose G proteins are single-point mutant proteins of theG protein of the I-4140 strain (cytoplasmic fragments of SEQ ID NO: 19and 20, respectively; cf. FIG. 19).

CNCM is the Collection Nationale de Cultures de Microorganismes;Institut Pasteur; 28, rue du Docteur Roux; F-75724 Paris Cedex 15;France. Said deposits have been made under the terms of the BudapestTreaty.

Variant Sequence a:

A variant sequence A of the invention comprises the sequence of SEQ IDNO: 14, i.e., the sequence x₁-x₂-x₃-x₄, wherein:

-   -   x₁ is any amino acid except E, and    -   x₂ is T or S or I, and    -   x₃ is any amino acid, and    -   x₄ is L or V.

This sequence is a particular PDZ-BS motif. It usually is located at theC-terminal end of the polypeptide, most usually this sequence is thesequence of the very last four C-terminal amino acids of thepolypeptide.

Preferably, said amino acid x₁ is Q.

Preferably, said amino acid x₂ is not I. Preferably it is T.

Preferably, said amino acid x₃ is R.

Preferably, said amino acid x₃ is L.

Preferably, the sequence of said PDZ-BS motif is the sequence of SEQ IDNO: 14, wherein:

-   -   x₁ is Q, and    -   x₂ is T or S or I, and    -   x₃ is R, and    -   x₄ is L or V.

Most preferably, the sequence of said PDZ-BS motif is the sequence ofSEQ ID NO: 14, wherein:

-   -   x₁ is Q, and    -   x₂ is T, and    -   x₃ is R, and    -   x₄ is L or V.

Most preferably, the sequence of said PDZ-BS motif is the sequence ofSEQ ID NO: 14, wherein:

-   -   x₁ is Q, and    -   x₂ is T or S or I, and    -   x₃ is R, and    -   x₄ is L.

More preferably, the sequence of said PDZ-BS motif is the sequence ofSEQ ID NO: 14, wherein:

-   -   x₁ is Q, and    -   x₂ is T, and    -   x₃ is R, and    -   x₄ is L,        i.e., it is the sequence of SEQ ID NO: 10 (cf. FIG. 16).

This QTRL sequence is the sequence of the PDZ-BS motif of the G proteinof the CVS-NIV strain, said strain being available from the CNCM underdeposit number I-4140, and/or of the protein coded by the plasmidavailable from the CNCM under deposit number I-2758 (recombinant E. colicontaining said plasmid), and/or of the protein of SEQ ID NO: 2 (cf.FIG. 13A).

Appropriate conditions for the cultivation of the recombinant E. colistrain containing the plasmid CNCM I-2758 coding for the G protein ofCVS-NIV comprise the incubation of said recombinant E. coli strain at37° C. on a standard LB-TYM growth medium (in the presence ofampicillin); cf. WO 03/048198.

Appropriate conditions for the propagation of the virus I-4140(recombinant rabies virus) comprise the incubation of said virus at 37°C. under 5% CO₂ with BHK-21 cells (sub-clone BSR) on a DMEM growthmedium containing glucose (e.g., 4.5 g/L), sodium pyruvate and glutamax(Invitrogen 31966047) and 5% FBS; cf. example 1 below.

Preferably, a variant sequence A of the invention is of 44 amino acidsand has the amino acid H or L, preferably H, at position 11 of its44aa-sequence.

Illustrative variant A polypeptides notably comprise the G protein ofthe recombinant rabies virus strain deposited on the 1 Apr. 2009 at theCNCM under deposit number I-4142 (whose G protein is a single-pointmutant protein of the G protein of I-4140; cf. FIG. 21), the cytoplasmicfragment thereof and the conservative cytoplasmic sub-fragments thereof.

The I-4142 strain differs from the CVS-NIV strain (CNCM I-4140) in thatits G protein has amino acid L at position 491 (position computed withrespect to the full length G protein), instead of H (cf. table 2 belowand FIG. 3D).

Appropriate conditions for the propagation of the virus I-4142(recombinant rabies virus) comprise the incubation of said virus at 37°C. under 5% CO₂ with BHK-21 cells (sub-clone BSR) on a DMEM growthmedium containing glucose (e.g., 4.5 g/L), sodium pyruvate and glutamax(Invitrogen 31966047) and 5% FBS; cf. example 1 below.

Illustrative variant A polypeptides notably comprise polypeptidescomprising the cytoplasmic fragment of SEQ ID NO: 19 or at least oneconservative sub-fragment thereof. The sequence of SEQ ID NO: 19 is anillustrative conservative variant of the sequence of SEQ ID NO: 6 (cf.FIG. 19). It stills has a positive neurite outgrowth effect, although ata lesser level than the sequence of SEQ ID NO: 6 (cf. FIG. 3D andassociated comments in the examples section).

Variant Sequence B:

A variant sequence B of the invention is of 44 amino acids.

It comprises the sequence of SEQ ID NO: 13 with x₁=E, i.e., the sequencex₁-x₂-x₃-x₄, wherein:

-   -   x₁ is E, and    -   x₂ is T or S or I (preferably not I, more preferably T), and    -   x₃ is any amino acid (preferably R), and    -   x₄ is L or V (preferably L).

This sequence is a particular PDZ-BS motif. It usually is located at theC-terminal end of the polypeptide, most usually this sequence is thesequence of the very last four C-terminal amino acids of thepolypeptide.

Preferably, said sequence of SEQ ID NO: 13 is ETRL (SEQ ID NO: 12).

In a variant sequence B of the invention, the amino acid, which is atposition 11, is not L. It is most preferably H.

Illustrative variant B polypeptides notably comprise the G protein ofthe recombinant rabies virus strain deposited on the 1 Apr. 2009 at theCNCM under deposit number I-4143 (whose G protein is a single-pointmutant protein of the G protein of I-4140; cf. FIG. 21), the cytoplasmicfragment thereof and the conservative cytoplasmic sub-fragments thereof.

The I-4143 strain differs from the CVS-NIV strain (CNCM I-4140) in thatits G protein has amino acid E at position 521 (position computed withrespect to the full length G protein), instead of Q (cf. table 2 belowand FIG. 3D).

Appropriate conditions for the propagation of the virus I-4143(recombinant rabies virus) comprise the incubation of said virus at 37°C. under 5% CO₂ with BHK-21 cells (sub-clone BSR) on a DMEM growthmedium containing glucose (e.g., 4.5 g/L), sodium pyruvate and glutamax(Invitrogen 31966047) and 5% FBS; cf. example 1 below.

Illustrative variant B polypeptides notably comprise polypeptidescomprising the cytoplasmic fragment of SEQ ID NO: 20 or at least oneconservative sub-fragment thereof. The sequence of SEQ ID NO: 20 is anillustrative conservative variant of the sequence of SEQ ID NO: 6 (cf.FIG. 19). It stills has a positive neurite outgrowth effect, although ata lesser level than the sequence of SEQ ID NO: 6 (cf. FIG. 3D andassociated comments in the examples section).

The G proteins of the I-4142 and the I-4143 strains induce asignificantly positive neurite outgrowth effect, although this effect issignificantly lower than the one of the G protein of the CVS-NIV strain(I-4140); cf. example 1 and FIG. 3D.

Therefore, each of the two amino acid positions contributes to theneurite outgrowth effect, i.e.:

-   -   the amino acid position, which, in the G protein of the CVS-NIV        strain, is position 491 (i.e., H in the G protein of the CVS-NIV        strain), and    -   the amino acid position, which, in the G protein of the CVS-NIV        strain, is the first amino acid of the PDZ-BS of the G protein,        i.e., position 521 in the G protein of the CVS-NIV strain (i.e.,        amino acid Q in the G protein of the CVS-NIV strain).        Alternative or Complementary Definition of a Polypeptide of the        Invention:

A polypeptide of the invention is a pro-neurite outgrowth polypeptide.

A polypeptide of the invention comprises a parent sequence and/or aconservative variant sequence (variant sequence A or B) and/or aconservative fragment sequence as above-defined.

Alternatively or complementarily, a polypeptide of the invention can bedefined as being the G protein of a rabies virus strain, or a variantsequence thereof which derives therefrom by one or several amino acidsubstitution(s) and/or addition(s) and/or deletion(s), or a fragmentsequence of such a G protein or variant G protein, more particularly acytoplasmic fragment or sub-fragment thereof, provided that saidpolypeptide has the function of inducing and/or stimulating neuriteoutgrowth, for example as below illustrated, more particularly neuriteoutgrowth from human pre-mitotic neurons, e.g., from the humanneuroblastoma cell line SH-SY5Y (ATCC CRL-2266; please see example 1below for illustrative experimental conditions and materials).

More particularly, a polypeptide of the invention can alternatively orcomplementarily be defined as being the G protein of a non-apoptoticrabies virus strain, or a variant sequence thereof which derivestherefrom by one or several amino acid substitution(s) and/oraddition(s) and/or deletion(s), or a fragment sequence of such a Gprotein or variant protein, more particularly a cytoplasmic fragment orsub-fragment thereof, provided that said variant sequence or saidfragment has retained the function of inducing and/or stimulatingneurite outgrowth, for example as below illustrated, more particularlyneurite outgrowth from human pre-mitotic neurons, e.g., from the humanneuroblastoma cell line SH-SY5Y (ATCC CRL-2266; please see example 1below for illustrative experimental conditions and materials).

Illustrative pro-neurite outgrowth proteins, which are not the G proteinof the CVS-NIV strain and/or which are other than the protein coded bythe plasmid available from the CNCM under deposit number 1-2758, and/orwhich are other than the protein of SEQ ID NO: 2, but which still aresuitable pro-neurite outgrowth polypeptides notably comprise the Gproteins of the recombinant rabies virus strains deposited on the 1 Apr.2009 at the CNCM under deposit numbers I-4142 and I-4143.

The I-4142 strain differs from the CVS-NIV strain (CNCM I-4140) in thatits G protein has amino acid L at position 491 (position computed withrespect to the full length G protein), instead of H (cf. table 2 belowand FIG. 3D).

The I-4143 strain differs from the CVS-NIV strain (CNCM I-4140) in thatits G protein has amino acid E at position 521 (position computed withrespect to the full length G protein), instead of Q (cf. table 2 belowand FIG. 3D).

The G proteins of the I-4142 and the I-4143 strains induce asignificantly positive neurite outgrowth effect, although this effect issignificantly lower than the one of the G protein of the CVS-NIV strain(I-4140).

Therefore, each of the two amino acid positions contributes to theneurite outgrowth effect, i.e.:

-   -   the amino acid position, which, in the G protein of the CVS-NIV        strain, is position 491 (i.e., H in the G protein of the CVS-NIV        strain), and    -   the amino acid position, which, in the G protein of the CVS-NIV        strain, is the first amino acid of the PDZ-BS of the G protein,        i.e., position 521 in the G protein of the CVS-NIV strain (i.e.,        amino acid Q in the G protein of the CVS-NIV strain).

Hence, illustrative pro-neurite outgrowth polypeptides comprise the Gprotein of the I-4142 strain and the G protein of the I-4143 strain, aswell as the fragments of these proteins, more particularly thecytoplasmic fragments thereof (SEQ ID NO: 19 and SEQ ID NO: 20 in FIG.19) and the cytoplasmic sub-fragments thereof, provided that thesesub-fragments have retained the function of inducing and/or stimulatingneurite outgrowth, for example as below illustrated, more particularlyneurite outgrowth from human pre-mitotic neurons, e.g., from the humanneuroblastoma cell line SH-SY5Y (ATCC CRL-2266; please see example 1below for illustrative experimental conditions and materials).

The invention more particularly relates to said polypeptide, for use instimulating and/or inducing neurite outgrowth, more particularly for usein the treatment and/or palliation and/or prevention of a disease,disorder or condition involving an insufficient or impaired neuriteoutgrowth, more particularly an insufficient or impaired neuritogenesis.

Preferably, the amino acid length of said polypeptide is of less than100 amino acids, more preferably of less than 90 amino acids.

The expression “non-apoptotic rabies virus G protein” is herein intendedaccording to its ordinary meaning in the field.

The PDZ-BS of a non-apoptotic rabies virus G protein is of SEQ ID NO:14.

A non-apoptotic rabies virus G protein can be additionally oralternatively characterized by the fact that its sequence is thesequence of a G protein of a rabies virus strain, and that it does nottrigger the apoptosis of human neurons.

Illustrative means to check that a candidate rabies virus G protein is anon-apoptogenic one are known to the person of ordinary skill in theart.

One of the means comprises checking that the rabies virus strain, whichcomprises this G protein, is a non-apoptotic strain when it infectsneurons, more particularly human neurons, such as SK-N-SH neuroblastomacell line (ATCC HTB11) or the SH-SY5Y neuroblastoma cell line (ATCCCRL-2266), preferably the SK-N-SH neuroblastoma cell line (ATCC HTB11).Such means are notably useful when a naturally-occurring rabies virusstrain comprising the candidate G protein is available for analysis.

Other means comprises genetically engineering cells to make them expressthe candidate G protein, infecting neuron cells with said expressedcandidate G protein, and determining that apoptosis is not induced bysaid infection.

An illustration of such means is described in Préhaud et al. 2003.

Illustrative genetically engineering cells comprise the transgenicJurkat cell line that is described in Préhaud et al. 2003, moreparticularly at page 10538 (cf. §“inducible transgenic cell lines”).

Illustrative neuron cells comprise the SK-N-SH neuroblastoma cell line(ATCC HTB11) or the SH-SY5Y neuroblastoma cell line (ATCC CRL-2266),preferably the SK-N-SH neuroblastoma cell line (ATCC HTB11).

Illustrative experimental conditions to make the genetically engineeringcells infect the neuron cells comprise those described in Préhaud et al.2003, more particularly at page 10538 (cf. §“inducible transgenic celllines”).

Detection that the rabies virus strain or the genetically engineeredcell does not induce the apoptosis of the neurons cells is within theambit of the person of average skill in the art. Illustrative meanscomprise those described in Préhaud et al. 2003 and Préhaud et al. 2005.

Illustrative means comprise detecting that no significant DNAfragmentation is induced, e.g., by Hoechst staining (cf. Préhaud et al.2003, more particularly at page 10538, §“Detection of nuclearfragmentation by Hoechst staining”), and/or by the TUNEL method (cf.Préhaud et al. 2003, more particularly at page 10538 (cf. §“Detection ofnuclear fragmentation by the TUNEL method”), and/or by DNAelectrophoresis (cf. Préhaud et al. 2005).

Additional or alternate illustrative means to detect that no significantDNA fragmentation is induced comprise the detection that caspase 8 isnot activated, e.g., following the procedure described in Préhaud et al.2003, more particularly at page 10538, §“Detection of caspaseactivation”), with the proviso that that the neuron cells used shouldthen contain caspase 8, which is the case of the SK-N-SH cell line.

Illustrative values of non significant apoptosis are shown in FIG. 5D ofPréhaud et al. 2003: cf. penultimate column with the heading“JrtTA-G-CVS”:

-   -   only 19% of apoptotic cells as measured by Hoechst staining;    -   only 4.7% of apoptotic cells as measured by assessment of the        activation of caspase 8.

The rabies virus strains which are attenuated strains (i.e., nonpathogenic, e.g., non pathogenic when injected intramuscularly inimmunocompetent mice), such as the attenuated ERA strain, the RV194-2strain, the AVO-1 strain, the SN10 strain, the SN-10-SAD strain, theSAG2 strain, are apoptogenic strains.

Those rabies virus strains, which are pathogenic, i.e., in vivoneurovirulent (such as the CVS-NIV strain) are non-apoptotic.

Therefore, a non-apoptogenic rabies virus strain is a pathogenic(neurovirulent) strain (e.g., pathogenic when injected intramuscularlyin immunocompetent mice).

The current knowledge is that:

-   -   when a non-apoptogenic rabies virus strain infect a human neuron        cell, the G protein it encodes accumulates in the cytoplasm of        said neuron cell under the form of perinuclear globular        structures, and is not diffusively distributed in the cytoplasm        of said neuron cell;    -   and that, to the contrary, when an apoptogenic rabies virus        infect a human neuron cell, the G protein it encodes does not        accumulate in the cytoplasm of said neuron cell under the form        of perinuclear globular structures, but is diffusively        distributed in the cytoplasm of said neuron cell.

As above-mentioned and below illustrated, a polypeptide of the inventioncan be the G protein of a rabies virus strain.

Preferably, said rabies virus G protein is:

-   -   i. the G protein of the CVS-NIV strain, said strain being        available from the CNCM under deposit number I-4140 (the deposit        date being the 1 Apr. 2009), and/or the protein coded by the        plasmid available from the CNCM under deposit number I-2758,        (the deposit date being the 30 Nov. 2001) and/or the protein of        SEQ ID NO: 2; or    -   ii. a variant protein of said rabies virus G protein of i.,        wherein said variant protein still is a rabies virus G protein,        and wherein the sequence of the PDZ-BS motif of said variant        rabies virus G protein still is the sequence of SEQ ID NO: 14.

Preferably, said rabies virus G protein is:

-   -   i. the G protein of the CVS-NIV strain which is available from        the CNCM under deposit number I-4140, and/or the protein coded        by the plasmid available from the CNCM under deposit number        I-2758, and/or the protein of SEQ ID NO: 2; or    -   ii. a variant protein of said rabies virus G protein of i.,        wherein:        -   said variant protein consists of a sequence which is at            least 95% identical (preferably at least 96%, more            preferably at least 97%, still more preferably at least            97.5%, even still more preferably at least 98%, most            preferably at least 98.5%, still most preferably at least            99%) to the sequence of said rabies virus G protein of i.            over the entire length of the sequence of said rabies virus            G protein of i., and        -   the sequence of the PDZ-BS motif of said variant rabies            virus G protein is the sequence of SEQ ID NO: 14.

Preferably, said rabies virus G protein is:

-   -   i. the G protein of the CVS-NIV strain which is available from        the CNCM under deposit number I-4140, and/or the protein coded        by the plasmid available from the CNCM under deposit number        I-2758, and/or the protein of SEQ ID NO: 2; or    -   ii. a variant protein of said rabies virus G protein of i.,        wherein said variant protein still is a rabies virus G protein,        and wherein:        -   said variant protein consists of a sequence which is at            least 95% identical (preferably at least 96%, more            preferably at least 97%, still more preferably at least            97.5%, even still more preferably at least 98%, most            preferably at least 98.5%, still most preferably at least            99%) to the sequence of said rabies virus G protein of i.            over the entire length of the sequence of said rabies virus            G protein of i., and        -   the sequence of the PDZ-BS motif of said variant rabies            virus G protein is the sequence of SEQ ID NO: 14.

Preferably, said rabies virus G protein is:

-   -   i. the G protein of the CVS-NIV strain which is available from        the CNCM under deposit number I-4140, and/or the protein coded        by the plasmid available from the CNCM under deposit number        I-2758, and/or the protein of SEQ ID NO: 2; or    -   ii. a variant protein of said rabies virus G protein of i.,        wherein said variant protein still is a rabies virus G protein,        and wherein said variant protein consists of a sequence which:        -   is at least 95% identical (preferably at least 96%, more            preferably at least 97%, still more preferably at least            97.5%, even still more preferably at least 98%, most            preferably at least 98.5%, still most preferably at least            99%) to the sequence of said rabies virus G protein of i.            over the entire length of the sequence of said rabies virus            G protein of i., and        -   has retained the PDZ-BS motif of SEQ ID NO: 14 of said            rabies virus G protein of i. and/or contains a PDZ-BS motif,            the sequence of which is QTRL (SEQ ID NO: 14 with x₁=Q;            x₂=T; x₃=R; x₄=L).

Preferably, the amino acid length of said variant rabies virus G proteinof ii. does not exceed the length of said rabies virus G protein of i.of more than 50 amino acids.

Preferably, the amino acid length of said variant rabies virus G proteinof ii. is no more than 50 amino acid lower than the length of saidrabies virus G protein of i., for example of the same length as thesequence of said rabies virus G protein of i.

More preferably, the amino acid length of said variant rabies virus Gprotein of ii. is comprised is at least the length of said rabies virusG protein of i minus 50 amino acids, and of at most the length of saidrabies virus G protein of i. plus 50 amino acids, for example of thesame length as the sequence of said rabies virus G protein of i.

Illustrative pro-neurite outgrowth rabies virus G proteins, which arenot the G protein of the CVS-NIV strain (I-4140) and/or which are otherthan the protein coded by the plasmid available from the CNCM underdeposit number I-2758, and/or which are other the protein of SEQ ID NO:2, but which still are suitable pro-neurite outgrowth rabies virus Gproteins notably comprise the G proteins of the recombinant rabies virusstrain deposited on the 1 Apr. 2009 at the CNCM under deposit numberI-4142.

The I-4142 strain differs from the CVS-NIV strain (CNCM I-4140) in thatits G protein has amino acid L at position 491 (position computed withrespect to the full length G protein), instead of H (cf. table 2 belowand FIG. 3D).

An illustrative fragment of the sequence of SEQ ID NO: 19 (cf. FIG. 19).

Other Sequences that May Additionally be Present:

As above-mentioned and below illustrated, a polypeptide of the inventioncomprises an amino acid sequence, which has an effect of pro-neuriteoutgrowth (and/or of neurite spouting and/or of axon growth and/or ofdendritic tree extension).

As above-mentioned and below illustrated, said pro-neurite outgrowthsequence can be defined as a parent sequence and/or a conservativevariant sequence (variant sequence A or B) and/or a conservativefragment sequence as above-defined.

As above-mentioned and below illustrated, said pro-neurite outgrowthsequence can, alternatively or complementarily, be defined as being thesequence of the G protein of a (non-apoptotic) rabies virus strain(e.g., the G protein of CNCM I-4140, I-4142 or I-4143), or a variantsequence thereof, which derives therefrom by one or several amino acidsubstitution(s) and/or addition(s) and/or deletion(s), or a fragmentsequence of such a G protein or variant G protein, more particularly acytoplasmic fragment or sub-fragment thereof.

Illustrative examples of such fragments of G protein or variant Gprotein notably comprise those fragments, which have retained thecytoplasmic domain and the transmembrane domain of said G protein orvariant G protein.

Such a transmembrane domain notably has the advantage of anchoring thecytoplasmic fragment in intracellular compartment(s) of the cells, moreparticularly in the endoplasmic reticulum and/or the Golgi membrane ofthe cells, whereby said cytoplasmic fragment exerts more efficiently itseffects of stimulation and/or induction of neurogeneration,neuroregeneration and neuroprotection. Please see examples 4 and 5below.

Hence, in addition to said pro-neurite outgrowth sequence, a polypeptideof the invention may further comprise a sequence, which anchors saidpolypeptide in the endoreticulum membrane and/or in the Golgi membraneof cells, more particularly of neuronal cells, more particularly ofhuman neuronal cells, said anchoring sequence being preferably at theN-terminal end of said pro-neurite outgrowth sequence, most preferablydirectly linked to the first amino acid at the N-terminal end of saidpro-neurite outgrowth sequence.

Hence, in addition to said pro-neurite outgrowth sequence, a polypeptideof the invention may further comprise an amino acid sequence, whichanchors said polypeptide in the endoreticulum membrane and/or in theGolgi membrane (preferably in the endoreticulum membrane and in theGolgi membrane) of cells, more particularly of neuronal cells, moreparticularly of human neuronal cells (e.g., the human neuroblastoma cellline SH-SY5Y cell line as described in the examples below, e.g., example4).

Said anchoring sequence preferably is at the N-terminal end of saidsequence of SEQ ID NO: 6 or variant sequence A or variant sequence B.Most preferably, said anchoring sequence is directly or indirectly,preferably directly, linked to the first amino acid at the N-terminalend of said sequence of SEQ ID NO: 6 or of said variant sequence A or ofsaid variant sequence B.

Such an anchoring sequence may e.g., be an amino acid sequence, whichhas the capacity of anchoring the cytoplasmic domain of SEQ ID NO: 6 inthe endoreticulum membrane and/or in the Golgi membrane of cells, moreparticularly of neuronal cells, more particularly of human neuronalcells (e.g., the human neuroblastoma cell line SH-SY5Y cell line asdescribed in the examples below, e.g., example 4).

Illustrative of such an anchoring sequence is the transmembrane domainsequence of the G protein of a rabies virus strain (e.g., anon-apoptotic rabies virus strain), preferably the transmembrane domainsequence of the G protein of rabies virus CVS-NIV, which is of SEQ IDNO: 23.

Hence, in addition of said sequence of SEQ ID NO: 6 or of said variantsequence A or of said variant sequence B, said polypeptide may furthercomprise, preferably at the N-terminal end of said sequence of SEQ IDNO: 6 or variant sequence A or variant sequence B, most preferablydirectly linked to the first amino acid at the N-terminal end of saidsequence of SEQ ID NO: 6 or of said variant sequence A or of saidvariant sequence B:

-   -   the sequence of the transmembrane domain of the G protein of a        rabies virus strain, more particularly of a non-apoptotic rabies        virus strain, or    -   a sequence, which differs from said sequence of rabies virus G        transmembrane domain by at least one amino acid substitution        and/or deletion and/or addition, preferably by amino acid        substitution(s) and/or deletion(s), more preferably by amino        acid substitution(s), but which has retained the capacity of        anchoring said polypeptide (more particularly the capacity of        anchoring the cytoplasmic domain of SEQ ID NO: 6—when said        anchoring sequence is directly linked to the first amino acid at        the N-terminal end of said sequence of SEQ ID NO: 6-), in the        endoreticulum membrane and/or in the Golgi membrane of cells,        more particularly of neuronal cells, more particularly of human        neuronal cells (e.g., the human neuroblastoma cell line SH-SY5Y        cell line as described in the examples below, e.g., example 4).

Hence, in addition to said pro-neurite outgrowth sequence, a polypeptideof the invention may further comprise, preferably at the N-terminal endof said sequence of SEQ ID NO: 6 or variant sequence A or variantsequence B, most preferably directly linked to the first amino acid atthe N-terminal end of said sequence of SEQ ID NO: 6 or of said variantsequence A or of said variant sequence B:

-   -   the sequence of the transmembrane domain of the G protein of a        rabies virus strain, more particularly of a non-apoptotic rabies        virus strain, or    -   a sequence, which differs from said sequence of rabies virus G        transmembrane domain by at least one amino acid substitution        and/or deletion and/or addition, preferably by amino acid        substitution(s) and/or deletion(s), more preferably by amino        acid substitution(s), but which has retained the capacity of        anchoring the polypeptide of SEQ ID NO: 6 (when said anchoring        sequence is directly linked to the first amino acid at the        N-terminal of said sequence of SEQ ID NO: 6), in the        endoreticulum membrane and/or in the Golgi membrane of cells,        more particularly of neuronal cells, more particularly of human        neuronal cells.

Preferred examples of such anchoring sequences notably comprise:

-   -   the sequence of the transmembrane domain of the G protein of a        rabies virus strain, more particularly of a non-apoptotic rabies        virus strain, still more particularly of the CVS-NIV strain, for        example the sequence of SEQ ID NO: 23, or    -   a variant sequence thereof, which is of 18 to 26 amino acids,        preferably of 18 to 22 amino acids, more preferably of 22 amino        acids, and which is at least 94% identical to said sequence of        SEQ ID NO: 23 over the shortest of the two sequences (i.e., over        the shortest of SEQ ID NO: 23 and of said variant sequence).

Such an anchoring sequence is particularly useful when it is linked to apro-neurite outgrowth sequence that is the sequence of SEQ ID NO: 6 orsaid variant sequence A, more particularly the sequence of SEQ ID NO: 6.

Hence, according to an embodiment of the invention, the amino acidsequence of a polypeptide of the invention comprises, or consists of thesequence of SEQ ID NO: 23 followed by (from N-terminal to C-terminalend) the sequence of SEQ ID NO: 6, wherein one to four amino acids,preferably one amino acid (e.g., M) is/are optionally present betweensaid sequence of SEQ ID NO: 23 and said sequence of SEQ ID NO: 6.

Illustrative of such a polypeptide is the polypeptide of SEQ ID NO: 25.

In addition to said pro-neurite outgrowth sequence, a polypeptide of theinvention may further comprise:

-   -   a fragment of the ectodomain of the G protein of a rabies virus        strain, more particularly of a non-apoptotic rabies virus        strain, still more particularly of the CVS-NIV strain, and/or    -   a signal peptide, this signal peptide being preferably the        signal peptide of the G protein of a rabies virus strain, more        particularly of a non-apoptotic rabies virus strain, still more        particularly of the CVS-NIV strain.

Said ectodomain fragment and/or signal peptide may be present in saidpolypeptide of the invention in addition to said anchoring sequence.

Preferably, when a signal peptide is comprised in said polypeptide, saidsignal peptide is the first component at the N-terminal end of thepolypeptide.

Preferably, when a signal peptide and an ectodomain fragment arecomprised in said polypeptide, said ectodomain fragment is comprised insaid polypeptide of the invention between said signal peptide and saidpro-neurite outgrowth sequence, preferably between said signal peptideand any possibly anchoring sequence that may be comprised in saidpolypeptide.

According to an embodiment of the invention, a polypeptide of theinvention comprises, or consists of, from its N-terminal end to itsC-terminal end:

said signal peptide (e.g., SEQ ID NO: 21), said anchoring sequence(e.g., SEQ ID NO: 23) and said pro-neurite outgrowth sequence.

According to another embodiment of the invention, a polypeptide of theinvention comprises, or consists of, from its N-terminal end to itsC-terminal end:

said signal peptide (e.g., SEQ ID NO: 21), said ectodomain fragment(e.g., the two amino acids GK), said anchoring sequence (e.g., SEQ IDNO: 23) and said pro-neurite outgrowth sequence.

Preferably, said ectodomain fragment is one to four amino acids, morepreferably of 2 amino acids.

Preferably, said ectodomain fragment is a fragment of the C-terminal endof said ectodomain, more preferably the last one to four, moreparticularly the last two amino acids at the C-terminal end of saidectodomain.

Hence, the amino acid sequence of a polypeptide of the invention mayfurther comprise, (directly or indirectly, preferably directly) linkedto the first amino acid at the N-terminal end of said anchoringsequence:

one to four amino acids, preferably two amino acids, more preferably oneto four amino acids from the C-terminal end of the ectodomain of the Gprotein of a rabies virus (e.g., a non-apoptotic rabies virus strain),still more preferably the last two amino acids of the C-terminal end ofthe ectodomain of the G protein of a rabies virus (e.g., a non-apoptoticrabies virus strain), for example amino acids GK.

Examples of such a polypeptide notably comprise the polypeptide of SEQID NO: 26 and the polypeptides, which comprise the sequence of SEQ IDNO: 26.

Hence, a polypeptide of the invention may further comprise a signalpeptide, preferably the signal peptide of the G protein of a rabiesvirus strain (e.g., a non-apoptotic rabies virus strain), said peptidesequence being preferably at the N-terminal end of said polypeptide(most preferably at the very N-terminal end of said polypeptide, i.e.,in N-term from any anchoring sequence that may be comprised in saidpolypeptide).

Examples of such a polypeptide notably comprise the polypeptide of SEQID NO: 27 and the polypeptides, which comprise the sequence of SEQ IDNO: 27.

Examples of Preferred Polypeptides:

Preferred polypeptides of the invention comprise polypeptides, which areof less than 100 amino acids, more preferably of as few as amino acidsas possible while still retaining a neurite outgrowth effect, morepreferably of less than 90 amino acids.

Preferred polypeptides of the invention comprise polypeptides, whichcomprise, or consist of the cytoplasmic fragments of said G proteins,most preferably the cytoplasmic fragment of SEQ ID NO: 6, SEQ ID NO: 19or SEQ ID NO: 20 (cf. FIG. 19).

Preferred polypeptides of the invention also comprise polypeptides,which comprise, or consist of the 11-44 sub-fragments of thesecytoplasmic fragments.

Preferred polypeptides of the invention comprise polypeptides, which, inaddition to said pro-neurite outgrowth sequence, comprise at least oneanchoring sequence as above-defined (e.g., the anchoring sequence of SEQID NO: 23; please see examples 4-6 below).

Nucleic Acids, Vectors and Cells:

The invention also relates to any nucleic acid, more particularly to anyDNA or RNA, which codes for a polypeptide of the invention, inaccordance with the universal genetic code, taking due account of itsdegeneracy.

As shown in FIG. 15, an illustrative nucleic acid that codes for thecytoplasmic fragment of SEQ ID NO: 6 (i.e., the cytoplasmic fragment ofthe CVS-NIV strain) is the nucleic acid of SEQ ID NO: 5.

As shown in FIG. 16, an illustrative nucleic acid that codes for thePDZ-BS of SEQ ID NO: 10 (i.e., the PDZ-BS of the CVS-NIV strain) is thenucleic acid of SEQ ID NO: 9.

The invention also relates to any nucleic acid vector, which comprisesat least one nucleic acid coding for a polypeptide of the invention.Said vector can be a transfection and/or expression vector.

Said expression vector may further comprise at least one expressionsignal and/or regulatory sequence upstream and/or downstream of saidnucleic acid, such as at least one promoter, or at least one enhancerand at least one promoter, upstream of said nucleic acid.

Said expression vector may for example be a plasmid.

Illustrative of such vectors is the plasmid available from the CNCMunder deposit number I-2758.

Interestingly, lentivirus vectors, AAV vectors, adeno-virus vectors andHerpes Simplex disabled virus vectors can be used.

Alternatively or complementarily, a nucleic acid of the invention can becoupled to, or otherwise associated with, a dendrimer such as liposomes,or a cationic polymer such as DEAE-dextrane or polyethylenimine, or aninert solid nanoparticle, such as a gold particle, or any othertransfection means that the person of average skill in the art may findappropriate.

The invention also relates to any cell, which comprises at least onepolypeptide of the invention, and/or at least nucleic acid of theinvention and/or at least one vector of the invention.

Said cell preferably is a cell, which has been genetically engineered tocomprise said polypeptide and/or nucleic acid and/or vector.

Said cell can be a eukaryotic cell, preferably a mammal cell, forexample a human cell or a non-human cell, most preferably a human cell.

Said cell can be a prokaryotic cell, preferably a bacterium, for exampleE. coli.

Said cell can be a virion, provided said virion does not induce anysignificantly deleterious or undesirable effect to the recipientsubject, more particularly to a human patient.

Preferably, said cell is not a human embryonic cell.

Uses:

The invention more particularly relates to said polypeptide and/ornucleic acid and/or vector and/or cell, for use in inducing and/orstimulating neurite outgrowth, more particularly in the treatment and/orpalliation and/or prevention of a disease, disorder or conditioninvolving an insufficient or impaired neuritogenesis, more particularlyan insufficient or impaired neurite outgrowth.

Said use in a non-immunogenic use.

Indeed, those rabies virus G proteins, which are appropriate to theinvention, can be defined as non-apoptotic G proteins.

Therefore, the polypeptide of the invention does not induce theformation of apoptotic bodies such as those that have been described inthe previously-published PCT international application WO 03/048198.

In accordance with the invention, the polypeptide of the invention isintended as an effector of neurite outgrowth (and/or of axon and/ordendrite development), e.g., for neuron differentiation from neuronprogenitors or neoplastic neurons, and/or for neuron regeneration ofimpaired neurons (both effects being obtained through stimulation ofneurite outgrowth).

Therefore, the polypeptide of the invention is not an immunogenic agentor adjuvant, or at the very least it is not used as an immunogenic agentor adjuvant and it is not used under conditions which would enable saidpolypeptide to act as an immunogenic agent or adjuvant.

More particularly, contrary to the G proteins of apoptotic rabies virusstrains, such as the G proteins of the attenuated ERA strain and of theother attenuated rabies virus strains, the polypeptide of the inventiondoes not raise a detectable humoral immune response when it isadministered to neurons.

The polypeptide of the invention is not used in a pharmaceuticalcomposition or drug, which would be an immunogenic composition or avaccine.

Accordingly, the polypeptide of the invention is preferably not coupledto, not fused to and not associated with any antigen, more particularlywith any antigen that would be a viral antigen, a tumor antigen, a cellantigen, the over-expression or alteration of which would lead to apathology such as a neuropathology.

Diseases, Disorders and Conditions:

The invention provides polypeptides, nucleic acids, vectors and cells.These products induce and/or stimulate neuritogenesis, more particularlyneurite outgrowth, still more particularly human neurite outgrowth.

A product of the invention preferably is a nucleic acid vector of theinvention, more particularly a plasmid of the invention.

More particularly, a product of the invention induces and/or stimulatesneuritogenesis, more particularly neurite outgrowth from pre-mitoticneurons, neoplastic neurons, neuron progenitors, as well as fromimpaired neurons.

Therefore, the invention relates to said product, for use as aneurogenerative and/or neuroregenerative and/or neuroprotective agent.

A product of the invention stimulates and/or induces neurite sproutingand/or axon growth and/or dendritic tree extension.

A product of the invention stimulates the activity of the growth cone.Furthermore, it prevents growth cone from collapsing upon contact with agrowth collapsing agent, such as LPA or oxidative stress (cf. example 1below).

A product of the invention consequently stimulates and/or inducessynaptogenesis and/or neurotransmission.

A product of the invention is an agent that inhibits the proliferationof neoplastic neurons, more particularly as a neuro-differentiatingagent.

A product of the invention is an agent that stimulates neuronaldevelopment and/or neuronal regeneration and/or axon growth and/ordendrite development and/or dendritic tree extension and/or neuronalplasticity and/or synaptogenesis and/or neurotransmission.

A product of the invention is an agent that prevents and/or inhibitsand/or blocks any kind of neurotoxicity which would lead to neuriteretraction and/or growth cone collapse.

A product of the invention is an agent that stimulates and/or inducesneurite outgrowth and/or growth cone activity after said neurite and/orcone has been in contact with a neurotoxic agent.

A product of the invention is an agent that prevents and/or inhibitsand/or blocks growth cone collapse and/or neurite retraction and/oraxodendritic damage or lesion and/or disruption of synaptic integrityand/or loss of neuron connectivity and/or damage to nerve endings and/orneurotransmission impairment.

A product of the invention is a means to induce and/or stimulate neuriteoutgrowth, which is notably useful:

-   -   in inducing neuron differentiation, for example in the treatment        and/or palliation and/or prevention of a neoplasm of the nervous        system, as well as    -   in regenerating impaired neurons, more particularly impaired        neurites, for example in the treatment and/or palliation and/or        prevention of a neurodegenerative disease, disorder or        condition, in the treatment and/or palliation and/or prevention        of microbial infections of the neurons, or in protecting neurons        from neurotoxic agents or oxidative stress.

Therefore, the invention relates to said product, for use in thetreatment and/or palliation and/or prevention of any disease, disorderor condition which involves an insufficient or impaired neuritogenesis,more particularly an insufficient or impaired neurite outgrowth.

Said disease, disorder or condition is alternatively or complementarilydefined as any disease, disorder or condition involving an unbalancedneuron cell cycle, wherein said neuron cell cycle is unbalanced:

-   -   either by excessive or undesired presence of pre-mitotic neurons        (more particularly, by insufficient neuron differentiation        and/or by excessive or undesired re-entry of post-mitotic        neurons into the neuron cell cycle, as is the case when a        neoplasm develops in the nervous system),    -   or by excessive or undesired neuron degeneration, more        particularly excessive or undesired neurite degeneration (as is        the case for a neurodegenerative disease, disorder or condition,        and for certain microbial infection of the neurons).

A product of the invention can be used in the treatment and/orpalliation and/or prevention of a disease, disorder or condition, whichalters the Central Nervous System (CNS) and/or the Peripheral NervousSystem (PNS), for example as a neurorestorative therapy and/orprevention and/or palliation.

The expression “Central Nervous System” or “CNS” is herein intended asmeaning the brain and (in case of a vertebrate animal) the spinal cord.

The peripheral nervous system (PNS) is the vast network of spinal andcranial nerves linking the body to the brain and spinal cord. The PNS issubdivided into the autonomic nervous system (sympathetic NS andparasympathetic NS) and the somatic nervous system. The PNS consists ofsensory neurons running from stimulus receptors to the CNS and motorneurons running from the CNS to the muscle and glands.

According to an embodiment of the invention, said disease, disorder orcondition is or involves a microbial infection of the nervous system,such as a bacterial and/or viral infection, more particularly a viralinfection.

Said viral infection can for example be a Herpes Virus Simplex (HSV)infection, more particularly a HSV type 1 infection (which leads toviral encephalopathy).

Said microbial infection can be a viral infection, which does not induceneuron apoptosis, such as a rabies virus infection.

Preferably, said microbial infection is a microbial infection thatinduces neuron apoptosis, such as poliomyelitis (cf. Blondel et al.,2005).

According to another embodiment of the invention, said disease, disorderor condition is or involves a non-viral disease, disorder or condition,more preferably a non-bacterial and non-viral disease, disorder orcondition, still more preferably a non-microbial disease, disorder orcondition.

According to an embodiment of the invention, said disease or disorder isor involves a neurodegenerative disease or disorder (for example, achronic neurodegenerative disease or disorder), such as non-viralencephalopathy, Alzheimer's disease, Parkinson's disease, ALS,Huntington disease, multiple sclerosis (MS) or rare genetic disease.

Preferably, said neurodegenerative disease or disorder is a non-viraldisease or disorder, more preferably a non-bacterial and non-viraldisease or disorder, still more preferably a non-microbial disorder.

According to an embodiment of the invention, said condition is orinvolves a neurodegenerative condition, such as aging.

Preferably, said neurodegenerative condition is a non-viral condition,more preferably a non-bacterial and non-viral condition, still morepreferably a non-microbial condition.

According to an embodiment of the invention, said disease, disorder orcondition is or involves a physical or ischemic injury of the nervoussystem, such as seizure, stroke, trauma, epilepsy.

Preferably, said physical or ischemic injury is a non-viral disease,disorder or condition, more preferably a non-bacterial and non-viraldisease, disorder or condition, still more preferably a non-microbialdisease, disorder or condition.

According to an embodiment of the invention, said disease, disorder orcondition involves the presence of a chemical neurotoxic agent and/or ofan oxidative stress.

Preferably, said disease, disorder or condition is a non-viral disease,disorder or condition, more preferably a non-bacterial and non-viraldisease, disorder or condition, still more preferably a non-microbialdisease, disorder or condition.

According to an embodiment of the invention, said disease is a neoplasm,more particularly a neoplasm which comprises neoplastic neurons.

The term “neoplasm” is herein more particularly intended as a malignantneoplasm, more particularly a cancer, still more particularly a tumor ora leukaemia, even still more particularly a tumor.

A product of the invention does not act as an immunogenic agent. Moreparticularly, a product of the invention does not act as an immunogenicagent, which would raise a humoral response against tumor antigens.

Preferably, a product of the invention cannot, on and of its own, act asan immunogenic agent. More particularly, a product of the inventioncannot, on and of its own, act as an immunogenic agent, which wouldraise a humoral response against tumor antigens.

A product of the invention acts as an antiproliferative agent.

A product of the invention induces and/or stimulates the neuriteoutgrowth from neoplastic neurons, thereby inducing and/or stimulatingthe differentiation of neoplastic neurons into mature neurons.

Said neoplasm can be a neoplasm of the CNS and/or PNS, preferably aganglioglioma, a brain tumor, a central neurocytoma, a medulloblastoma,an ependymoma, a teratoma, a neuroblastoma.

Preferably, said neoplasm is a non-viral neoplasm, more preferably anon-bacterial and non-viral neoplasm, still more preferably anon-microbial neoplasm

Any administration mode that the skilled person may find appropriate isencompassed by the present invention.

Depending on how the product of the invention is formulated, it canadministered by parenteral or enteral (e.g., oral) administration,preferably by parenteral administration, more preferably by parenteralinjection.

Pharmaceutical Composition or Drug; Method of Treatment:

The invention also relates to any pharmaceutical composition or drug,which comprises at least one polypeptide of the invention and/or atleast nucleic acid of the invention and/or at least one vector of theinvention and/or at least one cell of the invention.

The pharmaceutical composition or drug of the invention can be used forthe treatment and/or palliation and/or prevention of a disease, disorderor condition involving an insufficient or impaired neurite outgrowth asabove-described in more details.

The pharmaceutical composition or drug of the invention is not animmunogenic composition and is not a vaccine.

The pharmaceutical composition or drug of the invention may furthercomprise at least one pharmaceutically and/or physiologically acceptablevehicle (diluent, excipient, additive, pH adjuster, emulsifier ordispersing agent, preservative, surfactant, gelling agent, as well asbuffering and other stabilizing and solubilizing agent, etc.).

The pharmaceutical composition or drug of the invention does preferablynot contain any immune adjuvant.

Most preferably, the pharmaceutical composition or drug of the inventiondoes not comprise any antigen, more particularly any antigen that wouldbe a viral antigen, a tumor antigen, a cell antigen, the over-expressionor alteration of which would lead to a pathology such as aneuropathology.

The pharmaceutical composition or drug of the invention can for examplebe a liquid solution, suspension, emulsion, tablet, pill, capsule,sustained release formulation, or powder. Preferably, it is formulatedunder a form suitable for parenteral administration.

The invention also relates to a method of treatment of a subject, moreparticularly of a human being, in need thereof, which comprisesadministering to said subject or human being at least one polypeptide ofthe invention and/or at least nucleic acid of the invention and/or atleast one vector of the invention and/or at least one cell of theinvention as above-described.

In the application, the term “comprising”, which is synonymous with“including” or “containing”, is open-ended, and does not excludeadditional, unrecited element(s), ingredient(s) or method step(s),whereas the term “consisting of” is a closed term, which excludes anyadditional element, step, or ingredient which is not explicitly recited.

The term “essentially consisting of” is a partially open term, whichdoes not exclude additional, unrecited element(s), step(s), oringredient(s), as long as these additional element(s), step(s) oringredient(s) do not materially affect the basic and novel properties ofthe invention.

The term “comprising” (or “comprise(s)”) hence includes the term“consisting of” (“consist(s) of”), as well as the term “essentiallyconsisting of” (“essentially consist(s) of”). Accordingly, the term“comprising” (or “comprise(s)”) is, in the present application, meant asmore particularly encompassing the term “consisting of” (“consist(s)of”), and the term “essentially consisting of” (“essentially consist(s)of”).

Each of the relevant disclosures of all references cited herein isspecifically incorporated by reference. The following examples areoffered by way of illustration, and not by way of limitation.

Example 1 The Cytoplasmic Domain of the Protein G of the Non-ApoptoticCVS-NIV Rabies Virus Strain (Cytoplasmic Domain of G-CVS-NIV or of“G-Survival”) Induces Neurite Outgrowth, and the Induced NeuriteOutgrowth is Highly Resistant to Growth Cone Collapsing Agent (i.e.,LPA) and to Oxidative Stress (H₂O₂)

Neuroregeneration and neuroprotection are common milestones in fields aslarge as neurotoxicity, neurodegenerative diseases,trauma-seizure-stroke, encephalopathy or even more aging at large (cf.FIG. 1).

Immature human neurons, such as neuroblastoma cells, are able todifferentiate further if treated with the appropriate signalingmolecules. This is the case for the SH-SY5Y cells when they are treatedwith the cell permeable db-cAMP.

The inventors have developed a functional neuroprotection test, whichinvolves the use of a human neuroblastoma cell line, such as the humanneuroblastoma cell line SH-SY5Y. In this system, cells can be furtherdifferentiated and the outgrowth of the neurites can be monitored, whichallows testing for growth cone activity stimulating effectors. Moreover,the ability of elongated neurites to fight against retraction processescan be evaluated after drug treatment such as lysophosphatidic acid(LPA) or hydrogen peroxide (H₂O₂). Data gathered with such systems havebeen proved to be correlated with neurotoxicity analyses made in vivo.

The example below notably demonstrates that:

-   -   the G protein of a non-apoptotic (i.e., virulent) rabies virus        strain, i.e., the CVS-NIV strain, has a neurite outgrowth        promoting effect, i.e., some non-apoptotic rabies virus G        proteins induce and/or stimulate neuritogenesis;    -   this neurite outgrowth effect is sufficiently strong to protect        neurons from growth cone collapsing drug (LPA) and oxidative        stress (H₂O₂);    -   it is the cytoplasmic tail of the G protein of said        non-apoptotic CVS-NIV rabies virus strain, which is responsible        for this neurite outgrowth effect;    -   apoptotic rabies virus G proteins do not show this neurite        outgrowth effect; more particularly the G protein of the ERA        strain, which differs from the G protein of the CVS-NIV strain        by only six amino acids (and by only two amino acids in the        cytoplasmic tail) does not show this neurite outgrowth effect;    -   this is notably due to the fact that the PDZ-BS motif that is        contained in the cytoplasmic tail of said non-apoptotic rabies        virus G protein shows a single-point mutation compared to the        one of said apoptotic rabies virus G protein: the protein G of        the non-apoptotic CVS-NIV strain has amino acid Q at position        521 (position computed with respect the full length G protein of        the CVS-NIV strain, corresponding to position 41 in the        cytoplasmic fragment of said G protein), whereas G-ERA has E at        the same position;    -   the amino acid, which is at position 491 in the full length G        protein of the CVS-NIV strain (position 11 in the cytoplasmic        fragment of said G protein), also contributes to this neurite        outgrowth effect (amino acid H).        Material and Methods        Cells, Viruses and Molecular Clones

SH-SY5Y is a neuroblastoma cell line, which is available from theAmerican Type Culture Collection (ATCC; 10801 University Blvd.;Manassas, Va. 20110-2209; U.S.A.) under deposit number CRL-2266).

The original ERA and CVS strains of rabies virus (RABV) are availablefrom the ATCC under deposit number vr332 and vr959, respectively. TheERA and CVS strains, which have been used in this study, have beenpassaged in the inventors' laboratory for the last twenty years on BSRcells (a clone of Baby Hamster Kidney cells-clone 21, ATCC depositnumber BHK-21). These passages strains are the ERA-NIV strain and theCVS-NIV strain, respectively.

Throughout the examples and the figures of the application, CVS meansCVS-NIV and ERA means ERA-NIV (G protein CNCM I-2760; SEQ ID NO: 4),unless otherwise specified.

The CVS-NIV strain (rRABV CVS HQ) has been deposited at the CollectionNationale de Cultures de Microorganismes (CNCM) on the 1 Apr. 2009 underthe terms of the Budapest Treaty (CNCM; Institut Pasteur; 25, rue duDocteur Roux; F-75724 PARIS CEDEX 15; FRANCE). The CNCM deposit numberis I-4140.

The obtention and characterization of the molecular clonesrepresentative of the G protein of the ERA-NIV strain, i.e., ofG-ERA-NIV, and of the G protein of the CVS-NIV strain, i.e., ofG-CVS-NIV, have been described in Préhaud et al. 2003.

The sequences of these G proteins are also available under accessionnumber AF 406693 (for G-ERA-NIV) and AF 406694 (for G-CVS-NIV); cf. alsoFIGS. 13A and 13B.

The G protein of the CVS-NIV strain is also available from therecombinant E. coli strain deposited on the 30 Nov. 2001 at the CNCMunder the terms of the Budapest Treaty. The CNCM deposit number isI-2758. This recombinant E. coli comprises a plasmid (plasmidpRev-TRE-G-CVS; cf. WO 03/048198), which inducibly expresses the Gprotein of the CVS-NIV strain.

The G protein of the ERA strain is also available from the recombinantE. coli strain deposited on the 30 Nov. 2001 at the CNCM under the termsof the Budapest Treaty. The CNCM deposit number is I-2760. Thisrecombinant E. coli comprises a plasmid (plasmid pRev-TRE-G-ERA; cf. WO03/048198), which inducibly expresses the G protein of the ERA strain.

Appropriate conditions for the cultivation of the recombinant E. colistrain containing the plasmid CNCM I-2758 coding for the G protein ofthe rabies virus CVS-NIV strain and for the cultivation of therecombinant E. coli strain containing the plasmid CNCM I-2760 coding forthe G protein of the rabies virus ERA strain comprise the incubation ofsaid recombinant E. coli strain at 37° C. on a standard LB-TYM growthmedium (in the presence of ampicillin).

The recombinant rabies viruses (rRABV) were produced and isolatedfollowing the procedures described by Faul et al. 2008. rRABVs wereconstructed to harbor either the wild type “G survival” or “G death”sequences (G sequence of the CVS-NIV strain and of the ERA strain,respectively), or the cytoplasmic tail of G survival in an ERA genetic Ggene background, or the cytoplasmic tail of G death in a CVS-NIV geneticG gene background (cf. FIG. 2).

Recombinant rabies viruses were also produced, which derive from theCVS-NIV strain by mutations in the G coding sequence. These recombinantrabies viruses have a G protein, which differs from the G protein of theCVS-NIV strain by one or two amino acids, namely:

-   -   by the amino acid, which in the full length G protein sequence        of the CVS-NIV strain is at position 491 (position 11 in the        cytoplasmic fragment thereof); and/or    -   by the amino acid, which in the full length G protein sequence        of the CVS-NIV strain is at position 521 (position 41 in the        cytoplasmic fragment thereof).

More particularly, the following recombinant rabies virus strains wereproduced:

TABLE 2 Position 491 in the full Position 521 in the full length Gprotein of the length G protein of the virus (amino acid H in virus(amino acid Q in the G protein of the the G protein of the CVS-NIVstrain) CVS-NIV strain) rRABV CVS LE L E rRABV CVS LQ L Q rRABV CVS HE HErRABV CVS LE, rRABV CVS LQ and rRABV CVS HE have been deposited at theCNCM under the terms of the Budapest Treaty on the 1 Apr. 2009. The CNCMdeposit numbers are I-4141, I-4142 and I-4143, respectively.

Appropriate conditions for the propagation of the virus I-4141 or I-4142or I-4143 (recombinant rabies viruses) comprise the incubation of saidvirus at 37° C. under 5% CO₂ with BHK-21 cells (sub-clone BSR) on a DMEMgrowth medium containing glucose (e.g., 4.5 g/L), sodium pyruvate andglutamax (Invitrogen 31966047) and 5% FBS.

High-Throughput Neurite Outgrowth and Retraction Assays

SH-SY5Y human neuroblastoma cells are seeded on 24-well plates (CellBind plastic ware, Corning, USA) at a density of 40,000 cells per wellin non differentiating medium [DMEMF12 (Invitrogen, U.K.) with 20% FetalBovine Serum plus 1% Pen:Strep and 1% Glutamine], and cultured overnightat 37° C. 24 h post seeding non differentiation medium is replaced withdifferentiating medium [Neurobasal medium (Invitrogen, U.K.)supplemented with B27 supplement (Invitrogen, U.K.), 1% P/S, 1%Glutamine and 1 mM db-cAMP (dibutyril c-AMP is membrane permeable,Sigma)], and the cells are incubated for 6 h. Then, cells are mockinfected, infected with rRABV at a MOI 3 in differentiating medium.After 1 h of incubation, cells are washed once with differentiatingmedium, and after adding differentiating medium they are incubated for24 h at 37° C.

For natural differentiation, the same procedure is used but db-cAMP isomitted.

30 h post differentiation, the cells are fixed with 3% paraformaldehydein phosphate buffered saline (PBS) for 20 min at room temperature (RT)followed by treatment for 5 mn with 0.1% Triton-X-100 and 50% normalgoat serum (NGS) in PBS for 1 h at RT. Neuronal specific anti βIIItubulin Ab (Promega, France) and anti-RABV nucleocapsid Ab are used tostain the neurite processes and to reveal RABV infection respectively.Alternatively, cells are also stained with crystal violet whichpreserves the neurites processes.

Retraction assay is identical to the outgrowth assay as mentioned above,except for the addition of either 10 μM-30 μM or 50 μM LPA (Sigma, USA)in differentiating medium or 75 μM H₂O₂ (Sigma, USA), in B27 minus antioxidant containing differentiating medium. Cells are imaged using aLeica DM 5000B UV microscope equipped with a DC 300FX camera (×40 or ×20objectives) and analyzed using ImageJ 1.38X Software (Wayne Rasband,NIH, USA,) and its plug-in NeuronJ (Meijering et al. 2004). The averageneurite length per neuron is determined from triplicate experiments.

Detection of RABV Antigens by Flow Cytometry

SH-SY5Y cells were differentiated by treatment with db c-AMP andinfected with rRABVs 6 hours post differentiation. 24 hours postinfection, cells were harvested and treated for the detection by flowcytometry of either the RABV glycoprotein which reached the cytoplasmicmembrane or the total amount of G protein expressed in the infectedcells. The procedures followed have been described in Préhaud et al.2003.

Results and Discussion

G-CVS-NIV Cytoplasmic Tail (i.e., the Cytoplasmic Tail of the G Proteinof the CVS-NIV Strain) Contains a Molecular Signature Promoting NeuriteOutgrowth

In absence of infection, treatment with db-cAMP triggers elongation ofneurites (FIG. 3A, the two left panels). When cells are treated withdb-cAMP and infected with rRABV G-CVS-NIV, they exhibit longer neurites(191%, FIG. 3A, middle right panel). This result establishes that rRABVG-CVS-NIV promotes neurite outgrowth when the growth cone activity isstimulated by cAMP signaling.

In contrast, the length of the neurites is similar in between noninfected cells and rRABV G-ERA infected cells both treated with db-cAMP(FIG. 3B), which shows that G death expressing rRABV ERA has a silentphenotype for the neurite outgrowth assay. It is worth noting that thetime course of the experiment (i.e., 30 h post differentiation and 24hours post infection) is a time point earlier than the time point wherethe maximum level of apoptosis is detected in rRABV ERA infectedneuroblastoma cells (i.e., 48 h post infection).

By using rRABVs carrying the end swap of the G protein (cf. FIG. 2) asdescribed on FIG. 3C, the inventors show that this phenotype is totallytransferred by the cytoplasmic tail only. This result firmly establishesthat G-CVS-NIV cytoplasmic tail contains a molecular signature promotingneurite outgrowth when the elongation processes are initiated by thecAMP signaling pathway.

Furthermore, neurite outgrowth assays were performed with recombinantrabies virus, which differ from the CVS-NIV strain (rRABV G-CVS-NIV) byone or two amino acids in their respective G proteins.

More particularly, assays were performed with rRABV CVS LE, rRABV CVS LQand rRABV CVS HE, which express a G protein that differs from G-CVS-NIVby amino acid 491 and/or amino acid 521 (cf. table 2 above). The resultsof these assays are illustrated by FIG. 3D. In FIG. 3D, all thedifferences with respect to the control are statistically significant(p<0.005 Student's t test):

-   -   rRABV G-CVS HQ versus N.I.: p<0.0001;    -   rRABV G-CVS LQ versus N.I.: p=0.0002;    -   rRABV G-CVS HE versus N.I.: p=0.0111.        rRABV G-CVS-NIV has H and Q at positions 491 and 521 of its G        protein, respectively (positions computed with respect to the        full length G protein of CVS-NIV, these positions corresponding        to positions 11 and 41 in the cytoplasmic fragment of this G        protein, respectively).        rRABV CVS LQ has L and Q at these positions.        rRABV CVS HE has H and E at these positions.        rRABV CVS LE has L and E at these positions.

As illustrated by FIG. 3D, rRABV CVS LQ and rRABV CVS HE still show apositive neurite outgrowth effect, although at a significantly lowerextent that CVS-NIV (rRABV G-CVS-NIV) [Student's t test analysis: rRABVCVS HQ versus rRABV CVS LQ: p=0.0156; rRABV CVS HQ versus rRABV CVS HE:p=0.0009].

Therefore, position 491 and position 521 of the G protein of the CVSstrain (positions 11 and 41 in the cytoplasmic fragment) are bothtightly involved in the neurite outgrowth effect and position 521 has apredominant contribution thereto.

rRABV CVS LE did not induce any significantly positive neurite outgrowtheffect compared to the control (cf. FIG. 3C, where rRABV G-CVS CytoDeath=rRABV CVS LE).

G-CVS-NIV Cytoplasmic Tail is an Intrinsic Effector PromotingNeuritogenesis, which Works Synergistically with cAMP

The inventors investigated whether the expression of G-CVS-NIVcytoplasmic tail could on and of its own stimulate neuritogenesis.SH-SY5Y cells were infected with the different rRABV (cf. FIG. 2) andthe elongation of the neurites was monitored.

As observed in FIG. 4A, the expression of G-CVS-NIV or G-ERA-cytosurvival is sufficient to promote neurites outgrowth. These dataestablish that the cytoplasmic domain of G-CVS-NIV contains an intrinsicmolecular signature which signalized by an unknown mechanism the neuriteelongation processes. When the same experiment was undertaken with orwithout db-cAMP, it was observed that both G-CVS-NIV and cAMP mediatedneurites outgrowth work synergistically (cf. FIG. 4B).

Neuritogenesis Based Cytoplasmic Domain of G-CVS-NIV is Dependant of theMolecular Signature and not of the Amount of Expressed G Protein

The amount of rabies virus glycoprotein produced was analyzed verycarefully either by monitoring the total quantity of RABV antigenproduced in the infected db-cAMP treated SH-SY5Y cells, or the quantityof G-protein fully processed to the cytoplasmic membrane. The two rRABVsexpressing G-CVS-NIV or G-ERA have a similar amount of G-proteinexpressed in toto and at the cytoplasmic membrane (cf. FIG. 5). Thus,the survival phenotype, i.e., neurite outgrowth, is not linked to aphenomenon of quantity of available protein, but rather to specificdeterminant(s) present on the sequence of the cytoplasmic survivalpeptide.

Moreover, it is interesting to note that rRABV G-ERA cyto survivalexpress slightly less glycoprotein, but this virus still exhibits astrong survival phenotype (cf. FIGS. 3C, 4A). It means that as long asthe survival determinant(s) are present inside the infected cells, theamount of the peptide is not the major caveat and the neurites outgrowthis stimulated.

G-CVS-NIV Cytoplasmic Tail Confers Neuroprotection Against Growth ConeCollapsing Drug

LPA is a bioactive lipid acting as growth factor-like phospholipidswhich has been shown to exert diverse cellular functions that influencecell growth, motility, morphology and fate. On neuronal cells, LPAmediates growth cone collapsing and neurite retraction involving Rho andRho-kinase activation which drives the re-organization of the actomyosincytoskeleton.

The ability of the G protein cytoplasmic tail to protect against 10 μMLPA induced neurites retraction was investigated. When db-cAMPdifferentiated SH-SY5Y cells are treated with LPA, retraction of theneurites is observed (cf. FIG. 6A, left). The same effect is alsoobserved when cells are infected with rRABV G-ERA (cf. FIG. 6A, right).On the contrary, the long neurites detected on db-cAMP treated and rRABVG-CVS-NIV infected cells are totally preserved against treatment with agrowth cone collapsing agent (cf. FIG. 6A, middle). This resultestablishes that G-CVS-NIV possess some intrinsic neuroprotectiveproperties.

This protection is also noticed following infection with rRABVG-ERA-Cyto survival but not with rRABV G-CVS-Cyto death: cf. FIG. 6B.Therefore, the use of the end-swap mutants firmly establishes that theneuroprotection phenotype is linked to the expression of G-CVScytoplasmic domain.

G-CVS-NIV Cytoplasmic Tail Neuroprotection to LPA is Robust

LPA physiological range is usually around 1 μM. The inventors used ahigh dose of LPA, i.e., 10 μM, to validate the robustness of theG-CVS-NIV cytoplasmic tail conferred neuroprotection.

Sub-lethal doses of LPA up to 50 μM were tested in order to monitor theefficiency of this neuroprotection. SH-SY5Y cells were either noninfected or infected with rRABV G-CVS-NIV or rRABV G-ERA-Cyto survival.30 hours post differentiation, cells were treated with different dosesof LPA (cf. FIG. 7). Treatments of non infected cells with increasingamount of LPA drive a linear retraction of the length of the neurites(cf. FIG. 7, left). On the contrary, cells which have been infected withrRABV G-CVS-NIV do not exhibit any obvious collapsing of their neuriteswhatever is the dose of the LPA (cf. FIG. 7, middle). The same data areobserved for the cells infected with rRABV G-ERA-Cyto survival for adose of LPA up to 30 μM (cf. FIG. 7, right). The slight decreaseobserved for the highest dose of LPA (50 μM) might be relative to thelowest amount of G protein expressed by this rRABV. In this case, andfor a very high concentration of growth cone collapsing drug, therelative amount of G protein might become a limiting factor. In anycase, these data proved the very high efficiency of the G-CVS-NIVcytoplasmic tail conferred neuroprotection.

G-CVS-NIV Cytoplasmic Tail Confers Neuroprotection Against OxidativeStress

Based on the observations made above on the neuroprotection conferred byG-CVS-NIV cytoplasmic domain on LPA driven neurites retraction, theinventors asked the question whether this phenotype could be generalizedto other more ubiquitous agents.

Oxidative stress represents an important pathway leading to neuronaldegeneration. Oxidative stress has been implicated in manyneurodegenerative diseased but also in case of acute damage to the brainsuch as trauma, stroke and epilepsy. Therefore, the inventors undertooka study on the neurite retraction phenotypes after hydrogen peroxidetreatment. When differentiated cells are subjected to 75 μM H₂O₂, theirneurites shorten (cf. FIG. 8, first pair of bars at the left). The sameobservation is made when the cells have been previously infected withrRABV G-ERA (cf. FIG. 8, third pair of bars). To the contrary, noretraction of the neurites is observed when cells are infected withrRABV G-CVS-NIV (cf. FIG. 8, second pair of bars). Thus G-CVS-NIVexpression confers neuroprotection against an oxidative stress.

By using end swap mutants, we also showed that this neuroprotectiveproperty is linked to the expression of G-CVS-NIV cytoplasmic tail asdescribed on FIG. 8 (cf. fourth and fifth pairs of bars, relating torRABV G-CVS-Cyto death and rRABV G-ERA-Cyto survival, respectively; cf.FIG. 2 for the structure of these end-swap mutants).

the COOH Terminal PDZ-BS of the Cytoplasmic Domain of the Rabies Virus GProtein is Critically Involved in the Survival Phenotype Fate of theInfected Neuronal Cells.

Deletion of the last 4aa residues of the G-Cyto survival (survivalG-protein-delta) was sufficient to significantly reduce the survivalphenotype of the rRABV, as measured by its effect on the neuriteoutgrowth.

See for example FIG. 20, which shows the neurite outgrowth measured at 8hours post-infection, in the presence of db-cAMP, as induced by rRABVCVS-NIV Delta-PDZ-BS (CVS-NIV having amino acid H at position 491 andfrom which PDZ-BS has been deleted), compared to control (N.I.) and toG-CVS-NIV (CVS-NIV having amino acid H at position 491 and having itsPDZ-BS, i.e., QTRL in positions 521-524): the neurite outgrowth effectinduced by rRABV CVS-NIV Delta-PDZ-BS is significantly different fromthe one induced by the control (p=0.0002 Student's t test) and issignificantly different from the one induced by rRABV CVS-NIV (p=0.0003Student's t test).

The inventors notably demonstrate that:

-   -   protein G of CVS-NIV (G-CVS-NIV) is able to promote neurite        outgrowth (neuritogenesis) in a system where the growth cone        activity is stimulated by the c-AMP signaling pathway (delivered        as an external effector);    -   The cytoplasmic domain of G-CVS-NIV is responsible of this        phenotype;    -   The expression of the cytoplasmic tail of G-CVS-NIV is also        responsible for the stimulation of the neurite outgrowth in the        absence of c-AMP;    -   Both G-CVS-NIV and c-AMP work as synergistic effectors;    -   The phenotypes are linked to the molecular signature delivered        by the cytoplasmic tail of G-CVS-NIV, more particularly by those        amino acids which are at positions 491 and 521 in the full        length G-CVS-NIV protein (positions 11 and 41 in the cytoplasmic        fragment thereof), still more particularly by the amino acid,        which is at position 521 in the full length G-CVS-NIV protein        (position 41 in the cytoplasmic fragment thereof), and which is        part of the PDZ-BS;    -   The survival phenotype conferred by the G-CVS-NIV cytoplasmic        tail is highly resistant to treatments by growth cone collapsing        agent (i.e., LPA);    -   The survival phenotype conferred by the G-CVS-NIV cytoplasmic        tail is also resistant to oxidative stress (H₂O₂).

Example 2 The Cytoplasmic Domain of G-CVS-NIV Confers NeuroprotectionAgainst HSV-1 Cytophatic Effect

Herpes simplex virus type I encephalitis (HSVE) is the most commonsporadic fatal central nervous system infection in western countries andmanifest throughout the year in patients of all ages. HSVE develops whenherpes simplex virus type 1 infects brain tissues in a lytic:necroticmanner.

The ability of the G protein cytoplasmic tail to protect against thecytopathic effect of HSV-1 in cellulo was investigated.

Material and Methods

Neurite Outgrowth

As described in example 1 above.

HSV-1 Cytopathic Effect Determination

rRABV were produced as described in example 1 above.

rRABV was propagated in BSR cells (cf. Faul et al. 2008)

HSV-1 strain KOS (cf. Skare et al. 1975) was propagated in U373MG cells(ATCC HTB 17).

SH-SY5Y cells were mock infected or infected with rRABV indifferentiating medium minus db-c-AMP for 6 hours. Then, HSV-1 was addedat a multiplicity of infection of 3 (MOI 3) and the neurites outgrowthphenotypes were determined 24 hours later. In order to establish theeffect on neurite outgrowth of the sole HSV-1 infection, SH-SY5Y cellswere differentiated by db-c-AMP in differentiating medium for 6 hoursand the HSV-1 infection (MOI 3) was then realized.

Neurite outgrowth phenotype was determined as well 24 hours later.

Results and Discussion

SH-SY5Y neuroblastoma cells, which have been differentiated by treatmentwith db c-AMP, show a drastic retraction of their neurites (up to 79%,cf. FIG. 9A) after HSV-1 infection, which can eventually lead to thedeath of the cells.

To the contrary, when cells have been previously infected with rRABVG-CVS-NIV or rRABV G-ERA-Cyto Survival (cf. example 1 above), theyexhibit neurite outgrowth (cf. FIG. 9B) and are protected againstextensive neurite retraction (cf. FIG. 9C).

Example 3 The Cytoplasmic Domain of G-CVS-NIV ExhibitsAnti-Proliferative Properties for Neuroblastoma Cells

Neuroblastoma is the second most common solid tumor of childhoodaccounting for more than 13% of cancer death in children for the UnitedStates. Prognosis depends on the clinical stage of the disorder and theage of the child. Generally, most patients with neuroblastoma aretreated with therapeutic approaches that include surgery, radiation andcytotoxic chemotherapy.

Since human malignant neuroblastoma is characterized by poordifferentiation and uncontrolled proliferation of immature neuroblasts,pro-differentiative drugs such as all-trans-retinoic acid (ATRA) at highdoses have been used. The inventors have demonstrated that RABVG-CVS-NIV cytoplasmic tail is able to promote neurite outgrowth onSH-SY5Y human neuroblastoma cells (cf. the examples above), i.e., toinduce the differentiation of these cells. Therefore, the inventorswanted to answer the question of whether this differentiation propertywas efficient enough to control the proliferation of such cells inculture.

Material and Methods

MTT Assay

The assay is described by Sargent 2003. Briefly, cells, which have beenmock infected or infected with rRABV for 48 h in differentiating mediumwithout db c-AMP, are stained with the MTT solution for 3.5 hours at 37°C. with 5% CO₂. The mixture of medium and staining solution is thenremoved, the MTT crystals are dissolved with the dissolvent for one hourat room temperature under constant agitation. The assay is read at an ODof 550 nm. Each condition represents a n of 8. Statistical analysis wasdone with ANOVA tests.

Flow Cytometry

Cell counting and morphological changes were assessed by side andforward light scattering (SSC and FSC, respectively) as described inPréhaud et al. 2003. Each point represents the cells gathered during 1minute at low speed. Flow cytometry analyses were gated on a viablepopulation in order to exclude dead or necrotic cells which representless than 1% of the cell population at the 48 h time point.

Neurite Outgrowth

As described in example 1 above.

ATRA Treatment

All trans retinoic acid (ATRA) treatment of the neuroblastoma cells wasundertaken as already described by Préhaud et al. 2005.

Results and Discussion

In a first instance, SH-SY5Y cells were either non-treated or treatedwith ATRA at 5 μM or 10 μM in the culture medium. Cell proliferation waseither assayed by flow cytometry for viable cells or by cell countingvia an MTT assay. The data are presented on FIGS. 10A and 10B,respectively.

In each case, ATRA treatment of the cells leads to a slower growth ofthe neuroblasts with a decrease in proliferation varying in between 27%up to 55% depending on the test used to monitor cell growth. Thus,SH-SY5Y cells are effectively responding to the effect ofanti-proliferative drugs.

Therefore, SH-SY5Y cells were infected either with rRABV G-CVS-NIV orrRABV G-ERA and the proliferation of the cells (established by a MTTassay) as well as the outgrowth of the neurites were measured (cf. FIGS.11A and 11B). We found that the cell growth was inversely correlated tothe neurite outgrowth, establishing that the neurosurvival property ofG-CVS-NIV is associated by an intrinsic anti-proliferative phenotype onneuroblasts.

Neuroblastoma cells were infected in a second series of experiments witheither the parental rRABV G-CVS-NIV or rRABV G-ERA viruses as well asthe end swap mutants (rRABV G-CVS-Cyto death or rRABV G-ERA-Cytosurvival; cf. example 1 above and FIG. 2). Cell growth was measured byflow cytometry and MTT assay (cf. FIGS. 12A and 12B, respectively). Thedata showed that in each case when the cells are infected with neuritesoutgrowth promoting G-cyto survival tail viruses, the proliferation ofthe neuroblasts is affected varying from a lag in between 18.3% to 23%.Thus, G-Cyto survival possesses an intrinsic property to slow downneuroblasts cell growth.

Example 4 Experimental Demonstration that Pro-Survival Properties ofCyto-G are Conserved in the Expression Vector

This example demonstrates that, when it is expressed in the cell in theabsence of viral infection (polypeptides delivered at first as anexpression vector), the RABV cytoplasmic domain of the G protein of anon-apoptotioc rabies virus strain (Cyto-G) conserves the pro-survivalproperties (neuritogenesis and protection against retraction).

This example further investigates whether said survival propertiesdepend upon the size and upon the anchorage into the cytoplasmic orendoplasmic membrane.

Mutagenesis was undertaken by using oligonucleotides-PCR basedmutagenesis procedures successfully used to generate the rRABVs ofexample 1 (QuickChange Lightning site-directed mutagenesis kit, AgilentTechnologies; Stratagene product division; Catalog number 210518-12; kitused in accordance with the manufacture's recommendations; U.S. Pat.Nos. 7,176,004; 7,132,265; 7,045,328; 6,734,293; 6,489,150; 6,444,428;6,391,548; 6,183,997; 5,948,663; 5,932,419; 5,866,395; 5,789,166;5,545,552, and patents pending).

The oligonucleotides were designed for introducing respectively NheI andXmaI sites at the extremities of the constructs.

The mutagenic primers were:

GFullATG: (SEQ ID NO: 28) GGCCGCTAGCATGGTTCCTCAGGCTCTCCTGTTT GCytoATG:(SEQ ID NO: 29) GGCCGCTAGCATGAGAAGAGTCAATCGATCAGAACCT GendSTOP:(SEQ ID NO: 30) GGCCCCCGGGTCACAGTCTGGTCTGACCCCCACT mutTM.Cyto:(SEQ ID NO: 31) CCCCTTCTGGTTTTTCCATTGTGTTTTGGGGGGAAGTATGTATTACTGAGT

The mutants were screened by sequencing the inserts. By using thismethodology, the following three constructs (G-Full;G-[SP-(2aa)-TM-Cyto]; G-Cyto) were notably obtained:

G-Full: Accession Number AF 406694 (G Protein of CVS-NIV)

(SEQ ID NO: 2) MVPQALLFVPLLVFPLCFGKFPIYTIPDKLGPWSPIDIHHLSCPNNLVVEDEGCTNLSGFSYMELKVGYILAIKMNGFTCTGVVTEAETYTNFVGYVTTTFKRKHFRPTPDACRAAYNWKMAGDPRYEESLHNPYPDYHWLRTVKTTKESLVIISPSVADLDPYDRSLHSRVFPSGKCPGVAVSSTYCSTNHDYTIWMPENPRLGMSCDIFTNSRGKRASKGSETCGFVDERGLYKSLKGACKLKLCGVLGLRLMDGTWVAMQTSNETKWCPPDQLVNLHDFRSDEIEHLVVEELVRKREECLDALESIMTTKSVSFRRLSHLRKLVPGFGKAYTIFNKTLMEADAHYKSVRTWNEILPSKGCLRVGGRCHPHVNGVFFNGIILGPDGNVLIPEMQSSLLQQHMELLESSVIPLVHPLADPSTVFKDGDEAEDFVEVHLPDVHNQVSGVDLGLPNWGKYVLLSAGALTALMLIIFLMTCCRRVNRSEPTQHNLRGTGREVSVTPQSGKIISSWESHKSGGQTRL G-[SP-(2aa)-TM-Cyto]:

G-[SP-(2aa)-TM-Cyto] is a construct comprising (from N- to C-term):

-   -   the signal peptide of a G protein (e.g., the signal peptide of        CVS-NIV; SEQ ID NO: 21);    -   2 amino acids (e.g., from the ectodomain of a G protein, such as        from G-CVS-NIV);    -   the transmembrane domain of the G protein of a non-apoptotic        rabies virus strain (e.g., the transmembrane domain of        G-CVS-NIV; SEQ ID NO: 23);    -   the cytoplasmic domain of the G protein of a non-apoptotic        rabies virus strain (e.g., the cytoplasmic domain of G-CVS-NIV;        SEQ ID NO: 6).

The sequence of G-[SP-(2aa)-TM-Cyto] that was used in the presentexample is:

(SEQ ID NO: 27) MVPQALLFVPLLVFPLCFGGKYVLLSAGALTALMLIIFLMTCCRRVNRSEPTQHNLRGTGREVSVTPQSGKIISSWESHKSGGQTRL G-Cyto:

G-Cyto is a construct comprising (from N- to C-term): an amino acid(e.g., M) and the cytoplasmic domain of the G protein of a non-apoptoticrabies virus strain (e.g., the cytoplasmic domain of G-CVS-NIV; SEQ IDNO: 6).

The sequence of the G-Cyto that was used in the present example is:

(SEQ ID NO: 24) MRRVNRSEPTQHNLRGTGREVSVTPQSGKIISSWESHKSGGQTRL.

The sequences are shown on FIG. 22.

The sequence alignment is shown on FIG. 23.

A schematic representation of the three constructs is shown in FIG. 24.

Inserts were removed by NheI and XmaI digestion and were cloned in SAPdephosphorylated pCI-Neo derivative (Promega, France) by standardcloning procedures. E. coli XL1-blue cells (Stratagene, USA) weretransformed with the plasmid constructs. Plasmid DNA was extensivelypurified on Purelink columns (Invitrogen, U.K.) and the inserts weresequenced in order to verify the integrity of the sequence inserted inthe plasmid. Plasmid clones were used to nucleofect human neuroblastomacells [SH-SY5Y cells (ATCC CRL-2266), or Ntera cl2D1 cells (ATCCCRL-1973)] using the Amaxa GmbH electroporation technology (Amaxa®Nucleofector® kit V Catalog # VCA 1003 Lonza Germany following themanufacturer's recommendations). The expression of the G proteins wasassayed by flow cytometry as described in example 1.

For transient expression, the biological assays were undertaken 24 hpost nucleofection.

For stable expression, both nucleofected cell lines [SH-SY5Y or Nteracl2D1] were treated with G418 at 800 μg/ml over three weeks. Cell clonesresistant to G418 were isolated and frozen. The control cell lineconsists of cells, which have been nucleofected with pCI-Neo alonewithout any insert.

The neurite outgrowth assays and retraction assays were performed asdescribed in example 1.

Illustrative results are shown in FIGS. 25-28.

FIG. 25 illustrates the expression of a polypeptide comprising thecytoplasmic domain of a non-apoptotic rabies virus strain, moreparticularly the expression:

-   -   of the full length G protein of said rabies virus strain,    -   of a polypeptide, which comprises the transmembrane and        cytoplasmic domains of said G protein, but which does not        comprise the ectodomain of said G protein, and    -   of a polypeptide, which comprises the cytoplasmic domain of said        G protein, but which does not comprise the ectodomain and the        transmembrane domain of said G protein, compared to the        expression measured with the control plasmid.

As already illustrated, the G level expression is different and thebiological phenotypes are not correlated directly with the level ofexpression (see below and FIG. 5).

The transient expression of the cytoplasmic domain of the G protein of anon-apoptotic rabies virus strain—in the absence of viralinfection—induces and/or stimulates neurite outgrowth from humanneuroblastoma cells (FIG. 26). A transiently-expressed polypeptidecomprising the transmembrane domain in addition to the cytoplasmicdomain (but which does not comprise the ectodomain) of said G protein ismore efficient in inducing and/or stimulating neurite outgrowth than thetransiently-expressed full length G protein or than thetransiently-expressed cytoplasmic domain of said G protein (cytoplasmicdomain without the ectodomain and without the transmembrane domain)(FIG. 26).

The stable expression of the cytoplasmic domain of the G protein of anon-apoptotic rabies virus strain, or of polypeptides or proteinscomprising said domain—in the absence of viral infection—is feasible inhuman neuroblastoma cells (FIG. 27). A stably-expressed polypeptidecomprising the transmembrane domain in addition to the cytoplasmicdomain (but which does not comprise the ectodomain) of said G protein ismore efficient in inducing and/or stimulating neurite outgrowth than thestably-expressed full length G protein or than the stably-expressedcytoplasmic domain of said G protein (cytoplasmic domain without theectodomain and without the transmembrane domain) (FIG. 27). Thestably-expressed cytoplasmic domain of said G protein (cytoplasmicdomain without the ectodomain and without the transmembrane domain) isas efficient in inducing and/or stimulating neurite outgrowth as thestably-expressed full length G protein (FIG. 27).

The expression of a polypeptide comprising the cytoplasmic domain of anon-apoptotic rabies virus strain—in the absence of viralinfection—confers resistance to a neurotoxic agent (e.g., the growthcone collapsing drug LPA; (cf. FIG. 28, showing results obtained withthe G-(SP-[2a]-TM-Cyto) construct).

In the same experimental conditions, this batch of LPA is inducinggrowth cone collapsing as illustrated in FIGS. 6A, 6B, 7.

The transmembrane domain of G-(SP-[2a]-TM-Cyto) notably allows thepolypeptide expressed by this construct to be anchored in the Golgimembrane and/or the endoreticulum membrane of the cells, therebyincreasing the efficiency of the expressed polypeptide in inducingand/or stimulation neurogeneration, neuroregeneration andneuroprotection.

Hence, coupling the cytoplasmic domain of the G protein of anon-apoptotic rabies virus strain to such a membrane-anchoring sequenceincreases the neurogeneration, neuroregeneration and neuroprotectioneffects induced and/or stimulated by said cytoplasmic domain.

Example 5 Experimental Demonstration that the Cytoplasmic Domain of aNon-Apoptotic Rabies Virus Strain Possess Intrinsic Properties to DriveNeuronal Precursor (EC Cells) to Differentiation to Mature Neurons(Commitment)

The cytoplasmic domain of a non-apoptotic rabies virus strain, such asCyto-G (cf. example 4 above; e.g., SEQ ID NO: 6 or 24), promotes theoutgrowth of the neurites by such promoting the differentiation of theneuroblastoma cell line SH-SY5Y (cf. example 4 above).

Example 5 brings the experimental demonstration that this propertyreveals a wider ability of said cytoplasmic domain to promote and toorientate the commitment of the cell line toward the neuronaldifferentiation.

Production of pure post-mitotic human neurons from the embryoniccarcinoma cell line NTera 2cl.-D1 (ATCC CRL-1973) has been described inthe art, e.g., in Préhaud et al. 2005.

Constructs expressing the cytoplasmic domain of the G protein of anon-apoptotic rabies virus strain, as described in example 4 above, wereused, e.g.:

-   -   the G-Full construct containing the G protein of a non-apoptotic        rabies virus strain (G protein of SEQ ID NO: 2) as an insert,    -   the G-Cyto construct containing the cytoplasmic domain of a        non-apoptotic rabies virus strain (cytoplasmic domain of SEQ ID        NO: 6) as an insert, and    -   the G-(SP)-[2aa]-TM-Cyto construct, containing the signal        peptide of a non-apoptotic rabies virus strain (signal peptide        of SEQ ID NO: 21), two amino acids (from the C-terminal end of        the ectodomain of the G protein of a non-apoptotic rabies virus        strain, e.g., GK), the transmembrane domain of the G protein of        a non-apoptotic virus strain (transmembrane domain of SEQ ID        NO: 23) and the cytoplasmic domain of a non-apoptotic rabies        virus strain (cytoplasmic domain of SEQ ID NO: 6).

The ability of the G-expressing constructs to induce the differentiationof NTera 2-D1 cells into neurons was investigated.

Stable G-expressing NTera 2-D1 cells were processed by following theATRA-neurosphere like protocol as described Préhaud et al. 2005.

After the last antimitotic treatment and the two replicates, cells wereseeded either for accounting neurite tips (5 days post last replicate)or neuronal network architecture (50 days post last replicate).

Illustrative results are shown in FIGS. 29-31.

The expression of a polypeptide comprising the cytoplasmic domain of theG protein of a non-apoptotic rabies virus strain—in the absence of viralinfection—induces and/or stimulates the production of maturepost-mitotic human neurons by differentiation from neuroblastoma cells:cf. FIG. 29, illustrating the results obtained with the expression:

-   -   of the full length G protein of a non-apoptotic rabies virus        strain,    -   of a polypeptide comprising the transmembrane domain and the        cytoplasmic domain (but which does not comprise the ectodomain)        of said G protein,    -   of the cytoplasmic domain of said G protein (cytoplasmic domain        without the ectodomain and without the transmembrane domain),        compared to the control plasmid.

The polypeptide, which comprised the transmembrane domain in addition tothe cytoplasmic domain of the G protein of a non-apoptotic rabies virusstrain (but which does not comprise the ectodomain of said G protein) ismore efficient in inducing and/or stimulating said differentiation ofpost-mitotic human neurons than the full length G protein or than thecytoplasmic domain of said G protein (cytoplasmic domain without theectodomain and without the transmembrane domain); cf. FIG. 30.

Further to inducing and/or stimulating the production of maturepost-mitotic human neurons by differentiation from neuroblastoma cells,the expression of a polypeptide comprising the cytoplasmic domain of theG protein of a non-apoptotic rabies virus strain—in the absence of viralinfection—induces and/or stimulates the organisation of a network ofmature post-mitotic human neurons with long axons (cf. FIG. 31).

Example 6 Experimental Demonstration that the Cytoplasmic Domain of aNon-Apoptotic Rabies Virus Strain is Able to Promote Regeneration ofInjured Neurons

Injured neurons may have the possibility to regenerate depending ontheir origins and on the local environment. For example, neurons fromthe mammalian central nervous system have very limited regenerativecapacity, even though lesion to a peripheral process results inincreased regeneration of dorsal root ganglion neurons. It is clearlyestablished that intrinsic regeneration signals influence the success ofproper regeneration, some of them involving specific kinase pathway(Hammarlund et al. 2009).

The cytoplasmic domain of a non-apoptotic rabies virus strain, such asthe cytoplasmic domain of SEQ ID NO: 6 contained in the Cyto-G construct(cf. example 4 above), exhibits at least two interesting properties asfar as regenerative medicine is concerned. First, it promotes neuriteoutgrowth, which means that said cytoplasmic domain can stimulate theneuronal growth cone.

Secondly, it preserves neurites from retraction after treatment with LPAor H₂O₂, which means that said cytoplasmic domain can stimulate orreinforce molecular locks avoiding the collapsing of the growth cone.

In order to bring the experimental demonstration that these propertiesdrive the regeneration of an injured neuron, the phenomenon wasinvestigated in a cellular model. The NT2-N cell line (human neuronalcells), stably expressing the G-[SP-(2aa)-TM-Cyto] construct of example4 (construct containing SEQ ID NO: 27 as an insert), was chosen.

The pCI-Neo-NT2-N was used as control.

The NT2-N cell line was derived from differentiated Ntera-2cl.D1(NT2/D1; ATCC CRL-1973) cells as described in Préhaud et al. 2005,Cheung et al. 1999 and Paquet-Durand et al. 2003.

For scratch-induced assays, cells were seeded on poly-D-Lysin-laminincoated cell+(Sarstedt, Germany) 12 wells plastic ware, and were grownfor two days in order to recover completely after trypsinisation. Themedium was changed 10 h before scratching. Individual wounds were madewith an injection needle (26GX1/2″, 12-4.5). At least 10 scratching weremade on each individual well. Cells were fixed with PFA (4%) 3 days postwounding and stained with crystal violet solution. Cells are imagedusing a Leica DM 5000B microscope equipped with a DC 300FX camera (×20objective) and analysed using ImageJ 1.38X Software (Wayne Rasband, NIH,USA) and its plug-in NeuronJ. The average percentage of neuron inregeneration is determined from 8 experiments.

Illustrative results are shown in FIGS. 32-33.

The expression of a polypeptide, which comprises the cytoplasmic domainof the G protein of a non-apoptotic rabies virus strain, induces and/orstimulates the regeneration of wounded mature post-mitotic human neurons(cf. FIG. 32, illustrating the results obtained with the expression of apolypeptide comprising the transmembrane and cytoplasmic domains of theG protein of a non-apoptotic rabies virus strain).

The neurite regeneration thus induced after wounding is drastic, moreparticularly when the polypeptide comprise the transmembrane and thecytoplasmic domains of said G protein, but does not comprise theectodomain of said G protein (cf. FIG. 33, illustrating the resultsobtained with the expression of a polypeptide comprising thetransmembrane and cytoplasmic domains of the G protein of anon-apoptotic rabies virus strain).

Example 7

An infectious rRABV cDNA was generated from the CVS-N2c rabies virusstrain (Morimoto et al. 1999), a fixed pathogenic and non-apoptoticRABV.

The infectious rRABV cDNA thus generated (cN2C) is described in Schnellet al. 2010.

The gene coding for G-vir (the G protein of a non-apoptotic rabies virusstrain, such as the G protein of SEQ ID NO: 2) has been integrated inthis RABV cDNA. The infectious virus has been recovered as described inexample 1.

The recombinant virus has been injected to groups of 8 six-week-oldfemale C57B16 mice by intramuscular route with the number of virusparticles of rRABV G-vir to trigger fatal encephalitis in 80% of mice.Number of mice and experiments have been carefully evaluated to minimizethe animal contribution.

Disease progression has been monitored by scoring clinical signs andmortality (as described in Camelo et al. 2001). In the course ofinfection, groups of two or three mice have been perfused, and brainshave been removed, snap frozen and stored at −80° C. before beingprocessed for RNA extraction (half brain) and immunohistochemistry ormultiplex arrays (second half brain) in order to measure theneuroinvasiveness of the virus.

This model allows to further analyze different parameters of the braininnate immune response triggered by the rRABV strain.

BIBLIOGRAPHIC REFERENCES

-   Blondel et al., Poliovirus, pathogenesis of poliomyelitis, and    apoptosis, CTMI, 2005, 289, 25-56.-   Camelo et al. 2001 “Selective role for the p55 Kd TNF-α receptor in    immune unresponsiveness induced by an acute viral encephalitis”; J.    Neuroimmunol. 113: 95-108.-   Cheung et al. 1999; BioTechniques 26: 946-954.-   Faul et al. 2008; Virology 382: 226-238.-   Guigoni and Coulon 2002, Journal of NeuroVirology 8: 306-317.-   Hammarlund et al. 2009 “Axon regeneration requires a conserved MAP    kinase pathway” Science 323: 802-806.-   Kumar et al. 2007; Nature 448: 39-43 and associated Supplementary    Information (“Methods”).-   Lay et al. 2003; Ann. N. Y. Acad. Sci. 1010: 577-581.-   Loh et al. 2008; Cell Death and Differentiation 15: 283-298.-   Meijering et al. 2004; Cytometry Part A 58A: 167-176.-   Morimoto et al. 1999, Journal of Virology 73(1): 510-518.-   Paquet-Durand et al. 2003 Brain Res. Dev. Brain Res. 142: 161-167.-   Préhaud et al. 1988; Journal of Virology 62(1): 1-7.-   Préhaud et al. 2003; Journal of Virology 77(19): 10537-10547.-   Préhaud et al. 2005; Journal of Virology 79(20): 12893-12904.-   Sargent 2003; Recent Results in Cancer Research, vol. 161,    Springer-Verlag Berlin Heidelberg, pages 13-25-   Sarmento et al. 2005; Journal of NeuroVirology 11: 571-581.-   Schnell et al. 2010; Nature Reviews Microbiology 8(1): 51-65.-   Skare et al. 1975; J. Virol. 15:726-732 (Structure and function of    herpesvirus genomes. I. Comparison of five HSV-1 and two HSV-2    strains by cleavage of their DNA with Eco R I restriction    endonuclease).-   Ugolini 1995; The Journal of Comparative Neurology 356: 457-480.-   Ugolini 2008; Dodet B, Fooks A R, Müller T, Tordo N, and the    Scientific & Technical Department of the OIE (eds): Towards the    Elimination of Rabies in Eurasia. Dev. Biol. Basel, Karger, vol.    131, pp. 493-506.-   WO 03/048198; PCT international application in the name of Institut    Pasteur, Inv. Lafon et al., published on 12 Jun. 2003; as well as    its national/regional counterpart applications.-   Each of the relevant disclosures of all references cited herein is    specifically incorporated by reference.

What is claimed is:
 1. A product, which is: a polypeptide, wherein thesequence of said polypeptide consists of less than 100 amino acids,wherein said sequence of less than 100 amino acids comprises a sequenceconsisting of, in N- to C-terminal orientation: the sequence of thesignal peptide of the G protein of a rabies virus strain, the sequenceof a fragment of the ectodomain of the G protein of a rabies virusstrain, and the sequence of the transmembrane domain of the G protein ofa rabies virus strain, and wherein said sequence of less than 100 aminoacids further comprises: i. the sequence of SEQ ID NO: 6, or ii. asequence, which: is of 34 to 54 amino acids, is at least 94% identicalto said sequence of SEQ ID NO: 6 over the entire length of the shortestof the two sequences, comprises a PDZ-BS sequence, wherein said PDZ-BSsequence is: x₁ is Q, and x₂ is T or S or I, and x₃ is any amino acid,and x₄ is L or V (SEQ ID NO: 14), said sequence being referred to asvariant sequence A, or iii. a sequence, which: is of 44 amino acids, isat least 94% identical to said sequence of SEQ ID NO: 6, comprises aPDZ-BS sequence, wherein said PDZ-BS sequence is: x₁ is E, and x₂ is Tor S or I, and x₃ is any amino acid, and x₄ is L or V (SEQ ID NO: 13with x₁=E), and does not comprise the amino acid L at position 11, saidsequence being referred to as variant sequence B, or iv. a fragment ofsaid sequence of SEQ ID NO: 6 or of said variant sequence A or of saidvariant sequence B, wherein said fragment is of at least 34 amino acidsand has retained the PDZ-BS sequence of said sequence of SEQ ID NO: 6 orof said variant sequence A or of said variant sequence B, respectively,or a cell comprising said polypeptide, or a composition or a drug or apharmaceutical composition, comprising said polypeptide and/or saidcell.
 2. The product of claim 1, wherein the PDZ-BS sequence of saidvariant sequence A is QTRL (SEQ ID NO: 10).
 3. The product of claim 1,wherein said variant sequence A is of 44 amino acids and has the aminoacid H or L at position
 11. 4. The product of claim 1, wherein thePDZ-BS sequence of said variant sequence B is ETRL (SEQ ID NO: 12). 5.The product of claim 4, wherein said variant sequence B has the aminoacid H at position
 11. 6. The product of claim 1, wherein said fragmentis the fragment 11-44 from the sequence of SEQ ID NO:
 6. 7. The productof claim 1, wherein said polypeptide is or comprises the cytoplasmicfragment of the G protein of a rabies virus strain, or a sub-fragment ofsuch a cytoplasmic fragment.
 8. The product of claim 1, wherein saidrabies virus strain is a non-apoptotic rabies virus strain.
 9. Theproduct of claim 1, wherein said rabies virus strain is the straindeposited at the CNCM under I-4140, I-4142 or I-4143.
 10. The product ofclaim 1, wherein the sequence of said G protein transmembrane domain isat the N-terminal end of said sequence of i.-iv.
 11. The product ofclaim 1, wherein the sequence of said G protein transmembrane domain is:the sequence of SEQ ID NO: 23, or a sequence, which is of 18 to 26 aminoacids, and which is at least 94% identical to said sequence of SEQ IDNO: 23 over the shortest of the two sequences.
 12. The product of claim1, wherein said polypeptide comprises said sequence of SEQ ID NO: 6 orsaid variant sequence A.
 13. The product of claim 1, wherein the aminoacid sequence of said polypeptide comprises the sequence of SEQ ID NO:23 followed, in N- to C-terminal orientation, by the sequence of SEQ IDNO: 6, and wherein the number of amino acid(s), which is(are) betweensaid sequence of SEQ ID NO: 23 and said sequence of SEQ ID NO: 6, isfrom zero to four.
 14. The product of claim 1, wherein said polypeptidecomprises the sequence of SEQ ID NO:
 25. 15. The product of claim 1,wherein said polypeptide comprises the sequence of SEQ ID NO:
 26. 16.The product of claim 1, wherein said signal peptide is at the N-terminalend of said polypeptide.
 17. The product of claim 1, wherein saidpolypeptide is or comprises the sequence of SEQ ID NO:
 27. 18. Theproduct of claim 1, wherein the sequence of said fragment of G proteinectodomain consists of 1 to 4 amino acids.
 19. The product of claim 1,which stimulates or induces synaptogenesis and/or neurotransmissionand/or neuronal plasticity.
 20. The product of claim 1, which stimulatesor induces neuron generation and/or neuron regeneration and/or neuronprotection.
 21. The product of claim 1, which is not an immunogenicagent or adjuvant.
 22. A drug or a pharmaceutical composition, whichcomprises the polypeptide of claim 1 and/or the cell of claim 1, andwhich stimulates or induces neurite outgrowth and/or neurite spoutingand/or axon growth and/or dendritic tree extension from neurons.
 23. Adrug or a pharmaceutical composition, which comprises the polypeptide ofclaim 1 and/or the cell of claim 1, and which stimulates or inducesneurite outgrowth and/or neurite spouting and/or axon growth and/ordendritic tree extension from neurons, wherein said polypeptide and/orcell of claim 1 is an active principle for said stimulation and/orinduction, and/or wherein said polypeptide and/or cell of claim 1 is aneuro-differentiating agent.
 24. The drug or pharmaceutical compositionof claim 22, wherein said neurons are neurons that are impaired byneurodegeneration.
 25. The drug or pharmaceutical composition of claim22, wherein said neurons are neurons that are impaired by a microbialinfection of the nervous system and/or by a physical or ischemic injuryof the nervous system and/or by a chemical neurotoxic agent and/or by anoxidative stress.
 26. The drug or pharmaceutical composition of claim22, wherein said neurons are pre-mitotic neurons or neuron progenitors.